Opiate receptors

ABSTRACT

The invention provides isolated nucleic acid molecules, host cells that contain an isolated nucleic acid molecule, and substantially pure polypeptides. For example, the invention provides isolated nucleic acid molecules that encode polypeptides having mu3 opiate receptor activity, host cells that contain an isolated nucleic acid molecule that encodes a polypeptide having mu3 opiate receptor activity, and substantially pure polypeptides that have mu3 opiate receptor activity. In addition, the invention provides methods and materials for identifying mu3 opiate receptor agonists and antagonists.

CROSS-RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication Serial No. 60/270,479, filed Feb. 22, 2001 and U.S.Provisional Application Serial No. 60/336,677, filed Dec. 5, 2001.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0002] Funding for the work described herein was provided by the federalgovernment under NIDA number 5R24DA09010. Thus, the federal governmentmay have certain rights in the invention.

BACKGROUND

[0003] 1. Technical Field

[0004] The invention relates to opiate receptors. Specifically, theinvention relates to mu3 and mu4 opiate receptors as well as mu3 and mu4opiate receptor activation and inhibition.

[0005] 2. Background Information

[0006] Three general classes of cell surface opioid receptors (kappa,delta, and mu) have been described based on ligand specificity. Opioidreceptors exhibiting high binding specificity for morphine have beendesignated mu opioid receptors. Detailed analysis of mu opioid receptorsfrom various tissues has revealed the existence of multiple mu opioidreceptor subtypes. In fact, the cDNA encoding the mul opioid receptorsubtype has been identified. Oligonucleotides complementary to some, butnot all, exons of the mul opioid receptor can block the effects mediatedby the mu1 and mu2 receptor subtypes. Thus, the mu1 and mu2 opioidreceptor subtypes appear to share exon sequences, as would be expectedof splice variants. Supporting the idea of alternative splicing is thefinding of a single mu gene in human and mouse chromosomal DNA. Inaddition, a novel rat brain mu opioid receptor subtype, designatedrMOR1B, has been identified.

[0007] This receptor is identical to the rat mul opioid receptor at itsN-terminus but differs in its length and sequence at the C-terminus.Further, affinity studies demonstrated that the substrate specificity ofrMOR1B is similar to that of the rat mul opioid receptor, but rMOR1B ismore resistant to agonist-induced desensitization and has a differentexpression pattern in brain. The presence of another opiate receptor,designated mu3 opiate receptor, has been demonstrated pharmacologically.This mu3 opiate receptor is opioid peptide insensitive and opiatealkaloid selective. In addition, detailed binding analysis indicatesthat the mu3 opiate receptor is expressed by immune tissues (e.g., humanmonocytes and granulocytes).

SUMMARY

[0008] The invention relates to opiate receptors such as mu3 and mu4opiate receptors. Specifically, the invention provides isolated nucleicacid molecules that encode polypeptides having mu3 opiate receptoractivity, host cells that contain an isolated nucleic acid molecule thatencodes a polypeptide having mu3 opiate receptor activity, andsubstantially pure polypeptides that have mu3 opiate receptor activity.In addition, the invention provides methods and materials foridentifying mu3 opiate receptor agonists and antagonists.

[0009] The present invention is based on the discovery of nucleic acidthat encodes a polypeptide having mu3 opiate receptor activity. The term“mu3 opiate receptor” as used herein refers to a cell surfacepolypeptide that has a higher affinity for morphine than that for theopioid polypeptide [Tyr-D-Ala², Gly-N-Me-Phe⁴, Gly(ol)⁵]-enkephalin(DAMGO). The interaction of morphine with a mu3 opiate receptor caninduce changes in intracellular calcium concentration and nitric oxiderelease. Isolated nucleic acid molecules that encode a polypeptidehaving mu3 opiate receptor activity, host cells containing such isolatednucleic acid molecules, and substantially pure polypeptides having mu3opiate receptor activity are particularly useful to research scientistssince these materials allow scientists to explore, for example, theinteractions of morphine with the mu3 opiate receptor, the molecularmechanisms by which morphine induces intracellular calcium concentrationchanges, and the relationships of mu3 opiate receptors with other muopioid receptors. In addition, the methods and materials describedherein can be used to provide cells that are responsive to morphine. Forexample, cells can be transfected with a vector that directs expressionof a polypeptide having mu3 opiate receptor activity such that thosecells can respond to morphine stimulation.

[0010] In general, the invention features an isolated nucleic acidmolecule that encodes a polypeptide having mu3 opiate receptor activity.The isolated nucleic acid molecule can contain a nucleic acid sequencewith a length and a percent identity to the sequence set forth in SEQ IDNO: 1 over the length, where the point defined by the length and thepercent identity is within the area defined by points A, B, C, and D ofFIG. 1, where point A has coordinates (81, 100), point B has coordinates(81, 65), point C has coordinates (15, 65), and point D has coordinates(15, 100). The polypeptide can contain an amino acid sequence with alength and a percent identity to the sequence set forth in SEQ ID NO: 2over the length, where the point defined by the length and the percentidentity is within the area defined by points A, B, C, and D of FIG. 1,where point A has coordinates (26, 100), point B has coordinates (26,65), point C has coordinates (5, 65), and point D has coordinates (5,100). The isolated nucleic acid molecule can hybridizes underhybridization conditions to the sense or antisense strand of thesequence set forth in SEQ ID NO: 1 or 3. The isolated nucleic acidmolecule can contain the sequence set forth in SEQ ID NO: 4, 6, 8, or10.

[0011] In another embodiment, the invention features an isolated nucleicacid molecule that hybridizes under hybridization conditions to thesense or antisense strand of a nucleic acid that encodes a polypeptidehaving mu3 opiate receptor activity, where the isolated nucleic acidmolecule is at least 12 nucleotides in length, and where the isolatednucleic acid molecule does not hybridize to the sense or antisensestrand of the sequence set forth in SEQ ID NO: 12 or 13.

[0012] Another embodiment of the invention features an isolated nucleicacid molecule containing a nucleic acid sequence with a length and apercent identity to the sequence set forth in SEQ ID NO: 1 over thelength, where the point defined by the length and the percent identityis within the area defined by points A, B, C, and D of FIG. 1, wherepoint A has coordinates (81, 100), point B has coordinates (81, 65),point C has coordinates (15, 65), and point D has coordinates (15, 100).The isolated nucleic acid molecule can encode a polypeptide having mu3opiate receptor activity.

[0013] In another aspect, the invention features a cell containing anisolated nucleic acid molecule that encodes a polypeptide having mu3opiate receptor activity. The isolated nucleic acid molecule can containa nucleic acid sequence with a length and a percent identity to thesequence set forth in SEQ ID NO: 1 over the length, where the pointdefined by the length and the percent identity is within the areadefined by points A, B, C, and D of FIG. 1, where point A hascoordinates (81, 100), point B has coordinates (81, 65), point C hascoordinates (15, 65), and point D has coordinates (15, 100). Thepolypeptide can contain an amino acid sequence with a length and apercent identity to the sequence set forth in SEQ ID NO: 2 over thelength, where the point defined by the length and the percent identityis within the area defined by points A, B, C, and D of FIG. 1, wherepoint A has coordinates (26, 100), point B has coordinates (26, 65),point C has coordinates (5, 65), and point D has coordinates (5, 100).The isolated nucleic acid molecule can hybridize under hybridizationconditions to the sense or antisense strand of the sequence set forth inSEQ ID NO: 1 or 3. The isolated nucleic acid molecule can contain thesequence set forth in SEQ ID NO: 4, 6, 8, or 10.

[0014] In another embodiment, the invention features a cell containingan isolated nucleic acid molecule that hybridizes under hybridizationconditions to the sense or antisense strand of a nucleic acid thatencodes a polypeptide having mu3 opiate receptor activity, where theisolated nucleic acid molecule is at least 12 nucleotides in length, andwhere the isolated nucleic acid molecule does not hybridize to the senseor antisense strand of the sequence set forth in SEQ ID NO: 12 or 13.

[0015] Another aspect of the invention features a substantially purepolypeptide having mu3 opiate receptor activity. The polypeptide can beencoded by a nucleic acid sequence having a length and a percentidentity to the sequence set forth in SEQ ID NO: 1 over the length,where the point defined by the length and the percent identity is withinthe area defined by points A, B, C, and D of FIG. 1, where point A hascoordinates (81, 100), point B has coordinates (81, 65), point C hascoordinates (15, 65), and point D has coordinates (15, 100). Thepolypeptide can contain an amino acid sequence with a length and apercent identity to the sequence set forth in SEQ ID NO: 2 over thelength, where the point defined by the length and the percent identityis within the area defined by points A, B, C, and D of FIG. 1, wherepoint A has coordinates (26, 100), point B has coordinates (26, 65),point C has coordinates (5, 65), and point D has coordinates (5, 100).The polypeptide can be encoded by a nucleic acid molecule thathybridizes under hybridization conditions to the sense or antisensestrand of the sequence set forth in SEQ ID NO: 1 or 3. The polypeptidecan contain the sequence set forth in SEQ ID NO: 5, 7, 9, or 11.

[0016] Another aspect of the invention features a method for identifyinga mu3 opiate receptor agonist. The method includes (a) contacting a cellwith a test molecule, where the cell contains an isolated nucleic acidmolecule (e.g., exogenous nucleic acid molecule) that encodes apolypeptide having mu3 opiate receptor activity, and where the cellexpresses the polypeptide, and (b) determining whether or not the testmolecule induces, in the cell, a mu3 opiate receptor-mediated response.The determining step can include monitoring nitric oxide synthaseactivity in the cell. The monitoring nitric oxide synthase activity caninclude detecting nitric oxide release from the cell. A nitricoxide-specific amperometric probe can be used to detect the nitric oxiderelease. The determining step can include monitoring intracellularcalcium levels within the cell. A fluorescent ion indicator can be usedto monitor the intracellular calcium levels. The fluorescent ionindicator can be Fura-2. The determining step can contain monitoringnitric oxide synthase activity and intracellular calcium levels in thecell.

[0017] Another aspect of the invention features a method for identifyinga mu3 opiate receptor antagonist. The method includes (a) contacting acell with a test molecule and a mu3 opiate receptor agonist, where thecell contains an isolated nucleic acid molecule (e.g., exogenous nucleicacid molecule) that encodes a polypeptide having mu3 opiate receptoractivity, and where the cell expresses the polypeptide, and (b)determining whether or not the test molecule reduces or prevents, in thecell, a mu3 opiate receptor-mediated response induced by the mu3 opiatereceptor agonist. The mu3 opiate receptor agonist can contain morphineor dihydromorphine. The determining step can include monitoring nitricoxide synthase activity in the cell. The determining step can includemonitoring intracellular calcium levels within the cell.

[0018] Another aspect of the invention features an isolated nucleic acidmolecule containing a nucleic acid sequence with a length and a percentidentity to the sequence set forth in SEQ ID NO: 22 over the length,where the point defined by the length and the percent identity is withinthe area defined by points A, B, C, and D of FIG. 1, where point A hascoordinates (225, 100), point B has coordinates (225, 65), point C hascoordinates (15, 65), and point D has coordinates (15, 100).

[0019] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

[0020] Other features and advantages of the invention will be apparentfrom the following detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

[0021]FIG. 1 is a graph plotting length and percent identity with pointsA, B, C, and D defining an area indicated by shading.

[0022]FIG. 2 is a bar graph plotting the relative mu4 mRNA expressionlevel in mononuclear cells for the indicated treatments.

[0023]FIG. 3 is a bar graph plotting the relative mu4 mRNA expressionlevel in mononuclear cells treated with SNAP for the indicateddurations.

[0024]FIG. 4 is a bar graph plotting the relative mu4 mRNA expressionlevel in mononuclear cells treated with morphine for the indicateddurations.

DETAILED DESCRIPTION

[0025] The invention provides isolated nucleic acid molecules, hostcells that contain an isolated nucleic acid molecule, and substantiallypure polypeptides. In addition, the invention provides methods andmaterials for identifying mu3 opiate receptor agonists and antagonists.

[0026] Nucleic Acids

[0027] The term “nucleic acid” as used herein encompasses both RNA andDNA, including cDNA, genomic DNA, and synthetic (e.g., chemicallysynthesized) DNA. The nucleic acid can be double-stranded orsingle-stranded. Where single-stranded, the nucleic acid can be thesense strand or the antisense strand. In addition, nucleic acid can becircular or linear.

[0028] The term “isolated” as used herein with reference to nucleic acidrefers to a naturally-occurring nucleic acid that is not immediatelycontiguous with both of the sequences with which it is immediatelycontiguous (one on the 5′ end and one on the 3′ end) in thenaturally-occurring genome of the organism from which it is derived. Forexample, an isolated nucleic acid can be, without limitation, arecombinant DNA molecule of any length, provided one of the nucleic acidsequences normally found immediately flanking that recombinant DNAmolecule in a naturally-occurring genome is removed or absent. Thus, anisolated nucleic acid includes, without limitation, a recombinant DNAthat exists as a separate molecule (e.g., a cDNA or a genomic DNAfragment produced by PCR or restriction endonuclease treatment)independent of other sequences as well as recombinant DNA that isincorporated into a vector, an autonomously replicating plasmid, a virus(e.g., a retrovirus, adenovirus, or herpes virus), or into the genomicDNA of a prokaryote or eukaryote. In addition, an isolated nucleic acidcan include a recombinant DNA molecule that is part of a hybrid orfusion nucleic acid sequence.

[0029] The term “isolated” as used herein with reference to nucleic acidalso includes any non-naturally-occurring nucleic acid sincenon-naturally-occurring nucleic acid sequences are not found in natureand do not have immediately contiguous sequences in anaturally-occurring genome. For example, non-naturally-occurring nucleicacid such as an engineered nucleic acid is considered to be isolatednucleic acid. Engineered nucleic acid can be made using common molecularcloning or chemical nucleic acid synthesis techniques. Isolatednon-naturally-occurring nucleic acid can be independent of othersequences, or incorporated into a vector, an autonomously replicatingplasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), orthe genomic DNA of a prokaryote or eukaryote. In addition, anon-naturally-occurring nucleic acid can include a nucleic acid moleculethat is part of a hybrid or fusion nucleic acid sequence.

[0030] It will be apparent to those of skill in the art that a nucleicacid existing among hundreds to millions of other nucleic acid moleculeswithin, for example, cDNA or genomic libraries, or gel slices containinga genomic DNA restriction digest is not to be considered an isolatednucleic acid.

[0031] The term “exogenous” as used herein with reference to nucleicacid and a particular cell refers to any nucleic acid that does notoriginate from that particular cell as found in nature. Thus, allnon-naturally-occurring nucleic acid is considered to be exogenous to acell once introduced into the cell. It is important to note thatnon-naturally-occurring nucleic acid can contain nucleic acid sequencesor fragments of nucleic acid sequences that are found in nature providedthe nucleic acid as a whole does not exist in nature. For example, anucleic acid molecule containing a genomic DNA sequence within anexpression vector is non-naturally-occurring nucleic acid, and thus isexogenous to a cell once introduced into the cell, since that nucleicacid molecule as a whole (genomic DNA plus vector DNA) does not exist innature. Thus, any vector, autonomously replicating plasmid, or virus(e.g., retrovirus, adenovirus, or herpes virus) that as a whole does notexist in nature is considered to be non-naturally-occurring nucleicacid. It follows that genomic DNA fragments produced by PCR orrestriction endonuclease treatment as well as cDNAs are considered to benon-naturally-occurring nucleic acid since they exist as separatemolecules not found in nature. It also follows that any nucleic acidcontaining a promoter sequence and polypeptide-encoding sequence (e.g.,cDNA or genomic DNA) in an arrangement not found in nature isnon-naturally-occurring nucleic acid.

[0032] Nucleic acid that is naturally-occurring can be exogenous to aparticular cell. For example, an entire chromosome isolated from a cellof person X is an exogenous nucleic acid with respect to a cell ofperson Y once that chromosome is introduced into Y's cell.

[0033] The invention provides isolated nucleic acid molecules thatcontain a nucleic acid sequence having (1) a length, and (2) a percentidentity to an identified nucleic acid sequence over that length. Theinvention also provides isolated nucleic acid molecules that contain anucleic acid sequence encoding a polypeptide that contains an amino acidsequence having (1) a length, and (2) a percent identity to anidentified amino acid sequence over that length. Typically, theidentified nucleic acid or amino acid sequence is a sequence referencedby a particular sequence identification number, and the nucleic acid oramino acid sequence being compared to the identified sequence isreferred to as the target sequence. For example, an identified sequencecan be the sequence set forth in SEQ ID NO: 1.

[0034] A length and percent identity over that length for any nucleicacid or amino acid sequence is determined as follows. First, a nucleicacid or amino acid sequence is compared to the identified nucleic acidor amino acid sequence using the BLAST 2 Sequences (B12seq) program fromthe stand-alone version of BLASTZ containing BLASTN version 2.0.14 andBLASTP version 2.0.14. This stand-alone version of BLASTZ can beobtained from the State University of New York—Old Westbury campuslibrary as well as at Fish & Richardson's web site (www.fr.com) or theU.S. government's National Center for Biotechnology Information web site(www.ncbi.nlm.nih.gov). Instructions explaining how to use the B12seqprogram can be found in the readme file accompanying BLASTZ. B12seqperforms a comparison between two sequences using either the BLASTN orBLASTP algorithm. BLASTN is used to compare nucleic acid sequences,while BLASTP is used to compare amino acid sequences. To compare twonucleic acid sequences, the options are set as follows: -i is set to afile containing the first nucleic acid sequence to be compared (e.g.,C:\seq1.txt); -j is set to a file containing the second nucleic acidsequence to be compared (e.g., C:\seq2.txt); -p is set to blastn; -o isset to any desired file name (e.g., C:\output.txt); -q is set to −1; -ris set to 2; and all other options are left at their default setting.For example, the following command can be used to generate an outputfile containing a comparison between two sequences: C:\B12seq -ic:\seq1.txt -j c:\seq2.txt -p blastn -o c:\output.txt -q -1 -r 2. Tocompare two amino acid sequences, the options of B12seq are set asfollows: -i is set to a file containing the first amino acid sequence tobe compared (e.g., C:\seq1.txt); -j is set to a file containing thesecond amino acid sequence to be compared (e.g., C:\seq2.txt); -p is setto blastp; -o is set to any desired file name (e.g., C:\output.txt); andall other options are left at their default setting. For example, thefollowing command can be used to generate an output file containing acomparison between two amino acid sequences: C:\B12seq -i c:\seq1.txt -jc:\seq2.txt -p blastp -o c:\output.txt. If the target sequence shareshomology with any portion of the identified sequence, then thedesignated output file will present those regions of homology as alignedsequences. If the target sequence does not share homology with anyportion of the identified sequence, then the designated output file willnot present aligned sequences. Once aligned, a length is determined bycounting the number of consecutive nucleotides or amino acid residuesfrom the target sequence presented in alignment with sequence from theidentified sequence starting with any matched position and ending withany other matched position. A matched position is any position where anidentical nucleotide or amino acid residue is presented in both thetarget and identified sequence. Gaps presented in the target sequenceare not counted since gaps are not nucleotides or amino acid residues.Likewise, gaps presented in the identified sequence are not countedsince target sequence nucleotides or amino acid residues are counted,not nucleotides or amino acid residues from the identified sequence.

[0035] The percent identity over a determined length is determined bycounting the number of matched positions over that length and dividingthat number by the length followed by multiplying the resulting value by100. For example, if (1) a 1000 nucleotide target sequence is comparedto the sequence set forth in SEQ ID NO: 4, (2) the B12seq programpresents 200 nucleotides from the target sequence aligned with a regionof the sequence set forth in SEQ ID NO: 4 where the first and lastnucleotides of that 200 nucleotide region are matches, and (3) thenumber of matches over those 200 aligned nucleotides is 180, then the1000 nucleotide target sequence contains a length of 200 and a percentidentity over that length of 90 (i.e., 180÷200*100=90).

[0036] It will be appreciated that a single nucleic acid or amino acidtarget sequence that aligns with an identified sequence can have manydifferent lengths with each length having its own percent identity. Forexample, a target sequence containing a 20 nucleotide region that alignswith an identified sequence as follows has many different lengthsincluding those listed in Table 1.

[0037] Target Sequence:

[0038] Identified Sequence: 1                  20 AGGTCGTGTACTGTCAGTCA| || ||| |||| |||| | ACGTGGTGAACTGCCAGTGA

[0039] TABLE I Starting Ending Matched Percent Position Position LengthPositions Identity 1 20 20 15 75.0 1 18 18 14 77.8 1 15 15 11 73.3 6 2015 12 80.0 6 17 12 10 83.3 6 15 10  8 80.0 8 20 13 10 76.9 8 16  9  777.8

[0040] It is noted that the percent identity value is rounded to thenearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 is roundeddown to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 is rounded upto 78.2. It is also noted that the length value will always be aninteger.

[0041] The invention provides isolated nucleic acid molecules containinga nucleic acid sequence that has at least one length and percentidentity over that length as determined above such that the pointdefined by that length and percent identity is within the area definedby points A, B, C, and D of FIG. 1. In addition, the invention providesisolated nucleic acid molecules containing a nucleic acid sequence thatencodes a polypeptide containing an amino acid sequence that has atleast one length and percent identity over that length as determinedabove such that the point defined by that length and percent identity iswithin the area defined by points A, B, C, and D of FIG. 1. The pointdefined by a length and percent identity over that length is that pointon the X/Y coordinate of FIG. 1 where the X axis is the length and the Yaxis is the percent identity. Thus, the point defined by a nucleic acidsequence with a length of 200 and a percent identity of 90 hascoordinates (200, 90). For the purpose of this invention, any point thatfalls on point A, B, C, or D is considered within the area defined bypoints A, B, C, and D of FIG. 1. Likewise, any point that falls on aline that defines the area defined by points A, B, C, and D isconsidered within the area defined by points A, B, C, and D of FIG. 1.

[0042] It will be appreciated that the term “the area defined by pointsA, B, C, and D of FIG. 1” as used herein refers to that area defined bythe lines that connect point A with point B, point B with point C, pointC with point D, and point D with point A. Points A, B, C, and D candefine an area having any shape defined by four points (e.g., square,rectangle, or rhombus). In addition, two or more points can have thesame coordinates. For example, points B and C can have identicalcoordinates. In this case, the area defined by points A, B, C, and D ofFIG. 1 is triangular. If three points have identical coordinates, thenthe area defined by points A, B, C, and D of FIG. 1 is a line. In thiscase, any point that falls on that line would be considered within thearea defined by points A, B, C, and D of FIG. 1. If all four points haveidentical coordinates, then the area defined by points A, B, C, and D ofFIG. 1 is a point. In all cases, simple algebraic equations can be usedto determine whether a point is within the area defined by points A, B,C, and D of FIG. 1.

[0043] It is noted that FIG. 1 is a graphical representation presentingpossible positions of points A, B, C, and D. The shaded area illustratedin FIG. 1 represents one possible example, while the arrows indicatethat other positions for points A, B, C, and D are possible. In fact,points A, B, C, and D can have any X coordinate and any Y coordinate.For example, point A can have an X coordinate equal to the number ofnucleotides or amino acid residues in an identified sequence, and a Ycoordinate of 100. Point B can have an X coordinate equal to the numberof nucleotides or amino acid residues in an identified sequence, and a Ycoordinate less than or equal to 100 (e.g., 50, 55, 65, 70, 75, 80, 85,90, 95, and 99). Point C can have an X coordinate equal to a percent(e.g., 1, 2, 5, 10, 15, or more percent) of the number of nucleotides oramino acid residues in an identified sequence, and a Y coordinate lessthan or equal to 100 (e.g., 50, 55, 65, 70, 75, 80, 85, 90, 95, and 99).Point D can have an X coordinate equal to the length of a typical PCRprimer (e.g., 12, 13, 14, 15, 16, 17, or more) or antigenic polypeptide(e.g., 5, 6, 7, 8, 9, 10, 11, 12, or more), and a Y coordinate less thanor equal to 100 (e.g., 50, 55, 65, 70, 75, 80, 85, 90, 95, and 99).

[0044] Isolated nucleic acid molecules containing a nucleic acidsequence having a length and a percent identity to the sequence setforth in SEQ ID NO: 1 over that length are within the scope of theinvention provided the point defined by that length and percent identityis within the area defined by points A, B, C, and D of FIG. 1; wherepoint A has an X coordinate less than or equal to 81, and a Y coordinateless than or equal to 100; where point B has an X coordinate less thanor equal to 81, and a Y coordinate greater than or equal to 65; wherepoint C has an X coordinate greater than or equal to 15, and a Ycoordinate greater than or equal to 65; and where point D has an Xcoordinate greater than or equal to 15, and a Y coordinate less than orequal to 100. For example, the X coordinate for point A can be 81, 75,70, 65, 50, or less; and the Y coordinate for point A can be 100, 99,95, 90, 85, 80, 75, or less. The X coordinate for point B can be 81, 75,70, 65, 50, or less; and the Y coordinate for point B can be 65, 70, 75,80, 85, 90, 95, 99 or more. The X coordinate for point C can be 15, 16,17, 18, 19, 20, 25, 30, 40, 50, 75, or more; and the Y coordinate forpoint C can be 65, 70, 75, 80, 85, 90, 95, 99 or more. The X coordinatefor point D can be 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 75, or more;and the Y coordinate for point D can be 100, 99, 95, 90, 85, 80, 75, orless. In one embodiment, point A can be (81, 100), point B can be (81,95), point C can be (45, 95), and point D can be (45, 100).

[0045] Isolated nucleic acid molecules containing a nucleic acidsequence having a length and a percent identity to the sequence setforth in SEQ ID NO: 3 over that length are within the scope of theinvention provided the point defined by that length and percent identityis within the area defined by points A, B, C, and D of FIG. 1; wherepoint A has an X coordinate less than or equal to 262, and a Ycoordinate less than or equal to 100; where point B has an X coordinateless than or equal to 262, and a Y coordinate greater than or equal to65; where point C has an X coordinate greater than or equal to 45, and aY coordinate greater than or equal to 65; and where point D has an Xcoordinate greater than or equal to 12, and a Y coordinate less than orequal to 100. For example, the X coordinate for point A can be 262, 260,255, 250, 245, or less; and the Y coordinate for point A can be 100, 99,95, 90, 85, 80, 75, or less. The X coordinate for point B can be 262,260, 255, 250, 245, or less; and the Y coordinate for point B can be 65,70, 75, 80, 85, 90, 95, 99 or more. The X coordinate for point C can be45, 50, 60, 70, 80, 90, 100, 150, 200, or more; and the Y coordinate forpoint C can be 65, 70, 75, 80, 85, 90, 95, 99 or more. The X coordinatefor point D can be 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50,75, 100, or more; and the Y coordinate for point D can be 100, 99, 95,90, 85, 80, 75, or less. In one embodiment, point A can be (262, 100),point B can be (262, 95), point C can be (100, 95), and point D can be(100, 100).

[0046] Isolated nucleic acid molecules containing a nucleic acidsequence having a length and a percent identity to the sequence setforth in SEQ ID NO: 22 over that length are within the scope of theinvention provided the point defined by that length and percent identityis within the area defined by points A, B, C, and D of FIG. 1; wherepoint A has an X coordinate less than or equal to 225, and a Ycoordinate less than or equal to 100; where point B has an X coordinateless than or equal to 225, and a Y coordinate greater than or equal to65; where point C has an X coordinate greater than or equal to 45, and aY coordinate greater than or equal to 65; and where point D has an Xcoordinate greater than or equal to 12, and a Y coordinate less than orequal to 100. For example, the X coordinate for point A can be 225, 220,215, 210, 205, 200, 175, 150, or less; and the Y coordinate for point Acan be 100, 99, 95, 90, 85, 80, 75, or less. The X coordinate for pointB can be 225, 220, 215, 210, 205, 200, 175, 150, or less; and the Ycoordinate for point B can be 65, 70, 75, 80, 85, 90, 95, 99 or more.The X coordinate for point C can be 45, 50, 60, 70, 80, 90, 100, 150,200, or more; and the Y coordinate for point C can be 65, 70, 75, 80,85, 90, 95, 99 or more. The X coordinate for point D can be 12, 13, 14,15, 16, 17, 18, 19,20,25,30,40,50,75, 100, or more; and the Y coordinatefor point D can be 100, 99, 95, 90, 85, 80, 75, or less. In oneembodiment, point A can be (225, 100), point B can be (225, 95), point Ccan be (100, 95), and point D can be (100, 100).

[0047] Isolated nucleic acid molecules containing a nucleic acidsequence that encodes a polypeptide containing an amino acid sequencehaving a length and a percent identity to the sequence set forth in SEQID NO: 2 over that length are within the scope of the invention providedthe point defined by that length and percent identity is within the areadefined by points A, B, C, and D of FIG. 1; where point A has an Xcoordinate less than or equal to 26, and a Y coordinate less than orequal to 100; where point B has an X coordinate less than or equal to26, and a Y coordinate greater than or equal to 50; where point C has anX coordinate greater than or equal to 10, and a Y coordinate greaterthan or equal to 50; and where point D has an X coordinate greater thanor equal to 5, and a Y coordinate less than or equal to 100. Forexample, the X coordinate for point A can be 26, 25, 24, 23, 22, 21, 20,or less; and the Y coordinate for point A can be 100, 99, 95, 90, 85,80, 75, or less. The X coordinate for point B can be 26, 25, 24, 23, 22,21, 20, or less; and the Y coordinate for point B can be 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 99 or more. The X coordinate for point C can be10, 12, 14, 16, 17, 18, 20, or more; and the Y coordinate for point Ccan be 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or more. The Xcoordinate for point D can be 5, 6, 7, 8, 9, 10, 15, 20, or more; andthe Y coordinate for point D can be 100, 99, 95, 90, 85, 80, 75, orless. In one embodiment, point A can be (26, 100), point B can be (26,95), point C can be (10, 95), and point D can be (5, 100).

[0048] The invention also provides isolated nucleic acid molecules thatare at least about 12 bases in length (e.g., at least about 13, 14, 15,16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 100, 250, 500, 750, 1000, 1500,2000, 3000, 4000, or 5000 bases in length) and hybridize, underhybridization conditions, to the sense or antisense strand of a nucleicacid having the sequence set forth in SEQ ID NO: 1, 3, or 22. Thehybridization conditions can be moderately or highly stringenthybridization conditions. Such nucleic acid molecules can be moleculesthat do not hybridize to the sense or antisense strand of a nucleic acidhaving the sequence set forth in SEQ ID NO: 12 or 13.

[0049] For the purpose of this invention, moderately stringenthybridization conditions mean the hybridization is performed at about42° C. in a hybridization solution containing 25 mM KPO₄ (pH 7.4),5×SSC, 5×Denhart's solution, 50 μg/mL denatured, sonicated salmon spermDNA, 50% formamide, 10% Dextran sulfate, and 1-15 ng/mL probe (about5×10⁷ cpm/μg), while the washes are performed at about 50° C. with awash solution containing 2×SSC and 0.1% sodium dodecyl sulfate.

[0050] Highly stringent hybridization conditions mean the hybridizationis performed at about 42° C. in a hybridization solution containing 25mM KPO₄ (pH 7.4), 5×SSC, 5×Denhart's solution, 50 μg/mL denatured,sonicated salmon sperm DNA, 50% formamide, 10% Dextran sulfate, and 1-15ng/mL probe (about 5×10⁷ cpm/μg), while the washes are performed atabout 65° C. with a wash solution containing 0.2×SSC and 0.1% sodiumdodecyl sulfate.

[0051] Isolated nucleic acid molecules within the scope of the inventioncan be obtained using any method including, without limitation, commonmolecular cloning and chemical nucleic acid synthesis techniques. Forexample, PCR can be used to obtain an isolated nucleic acid moleculecontaining a nucleic acid sequence sharing similarity to the sequencesset forth in SEQ ID NO: 1, 3, or 22. PCR refers to a procedure ortechnique in which target nucleic acid is amplified in a manner similarto that described in U.S. Pat. No. 4,683,195, and subsequentmodifications of the procedure described therein. Generally, sequenceinformation from the ends of the region of interest or beyond are usedto design oligonucleotide primers that are identical or similar insequence to opposite strands of a potential template to be amplified.Using PCR, a nucleic acid sequence can be amplified from RNA or DNA. Forexample, a nucleic acid sequence can be isolated by PCR amplificationfrom total cellular RNA, total genomic DNA, and cDNA as well as frombacteriophage sequences, plasmid sequences, viral sequences, and thelike. When using RNA as a source of template, reverse transcriptase canbe used to synthesize complimentary DNA strands.

[0052] Isolated nucleic acid molecules within the scope of the inventionalso can be obtained by mutagenesis. For example, an isolated nucleicacid containing a sequence set forth in SEQ ID NO: 1, 3, or 22 can bemutated using common molecular cloning techniques (e.g., site-directedmutagenesis). Possible mutations include, without limitation, deletions,insertions, and substitutions, as well as combinations of deletions,insertions, and substitutions.

[0053] In addition, nucleic acid and amino acid databases (e.g.,GenBank®) can be used to obtain an isolated nucleic acid molecule withinthe scope of the invention. For example, any nucleic acid sequencehaving some homology to a sequence set forth in SEQ ID NO: 1, 3, or 22,or any amino acid sequence having some homology to a sequence set forthin SEQ ID NO: 2 can be used as a query to search GenBank®.

[0054] Further, nucleic acid hybridization techniques can be used toobtain an isolated nucleic acid molecule within the scope of theinvention. Briefly, any nucleic acid molecule having some homology to asequence set forth in SEQ ID NO: 1, 3, or 22 can be used as a probe toidentify a similar nucleic acid by hybridization under conditions ofmoderate to high stringency. Once identified, the nucleic acid moleculethen can be purified, sequenced, and analyzed to determine whether it iswithin the scope of the invention as described herein.

[0055] Hybridization can be done by Southern or Northern analysis toidentify a DNA or RNA sequence, respectively, that hybridizes to aprobe. The probe can be labeled with a biotin, digoxygenin, an enzyme,or a radioisotope such as ³²P. The DNA or RNA to be analyzed can beelectrophoretically separated on an agarose or polyacrylamide gel,transferred to nitrocellulose, nylon, or other suitable membrane, andhybridized with the probe using standard techniques well known in theart such as those described in sections 7.39-7.52 of Sambrook et al.,(1989) Molecular Cloning, second edition, Cold Spring harbor Laboratory,Plainview, N.Y. Typically, a probe is at least about 20 nucleotides inlength. For example, a probe corresponding to a 20 nucleotide sequenceset forth in SEQ ID NO: 1 or 3 can be used to identify an identical orsimilar nucleic acid. In addition, probes longer or shorter than 20nucleotides can be used.

[0056] The invention provides isolated nucleic acid molecules thatcontain the entire nucleic acid sequence set forth in SEQ ID NO: 1, 3,4, 6, 8, 10, 17, 21, 22, or 23. In addition, the invention providesisolated nucleic acid molecules that contain a portion of the nucleicacid sequence set forth in SEQ ID NO: 1, 3, or 22. For example, theinvention provides an isolated nucleic acid molecule that contains a 15nucleotide sequence identical to any 15 nucleotide sequence set forth inSEQ ID NO: 1, 3, or 22 including, without limitation, the sequencestarting at nucleotide number 1 and ending at nucleotide number 15, thesequence starting at nucleotide number 2 and ending at nucleotide number16, the sequence starting at nucleotide number 3 and ending atnucleotide number 17, and so forth. It will be appreciated that theinvention also provides isolated nucleic acid molecules that contain anucleotide sequence that is greater than 15 nucleotides (e.g., 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides)in length and identical to any portion of the sequence set forth in SEQID NO: 1, 3, or 22. For example, the invention provides an isolatednucleic acid molecule that contains a 25 nucleotide sequence identicalto any 25 nucleotide sequence set forth in SEQ ID NO: 1, 3, or 22including, without limitation, the sequence starting at nucleotidenumber 1 and ending at nucleotide number 25, the sequence starting atnucleotide number 2 and ending at nucleotide number 26, the sequencestarting at nucleotide number 3 and ending at nucleotide number 27, andso forth. Additional examples include, without limitation, isolatednucleic acid molecules that contain a nucleotide sequence that is 50 ormore nucleotides (e.g., 100, 150, 200, 250, 300, 350, or morenucleotides) in length and identical to any portion of the sequence setforth in SEQ ID NO: 1, 3, or 22.

[0057] In addition, the invention provides isolated nucleic acidmolecules that contain a variation of the nucleic acid sequence setforth in SEQ ID NO: 1, 3, or 22. For example, the invention provides anisolated nucleic acid molecule containing a nucleic acid sequence setforth in SEQ ID NO: 1, 3, or 22 that contains a single insertion, asingle deletion, a single substitution, multiple insertions, multipledeletions, multiple substitutions, or any combination thereof (e.g.,single deletion together with multiple insertions). The invention alsoprovides isolated nucleic acid molecules that contain a variant of aportion of the nucleic acid sequence set forth in SEQ ID NO: 1, 3, or 22as described herein.

[0058] The invention provides isolated nucleic acid molecules thatcontain a nucleic acid sequence that encodes the entire amino acidsequence set forth in SEQ ID NO: 2. In addition, the invention providesisolated nucleic acid molecules that contain a nucleic acid sequencethat encodes a portion of the amino acid sequence set forth in SEQ IDNO: 2. For example, the invention provides isolated nucleic acidmolecules that contain a nucleic acid sequence that encodes a 5 aminoacid sequence identical to any 5 amino acid sequence set forth in SEQ IDNO: 2 including, without limitation, the sequence starting at amino acidresidue number 1 and ending at amino acid residue number 5, the sequencestarting at amino acid residue number 2 and ending at amino acid residuenumber 6, the sequence starting at amino acid residue number 3 andending at amino acid residue number 7, and so forth. It will beappreciated that the invention also provides isolated nucleic acidmolecules that contain a nucleic acid sequence that encodes an aminoacid sequence that is greater than 5 amino acid residues (e.g., 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, ormore amino acid residues) in length and identical to any portion of thesequence set forth in SEQ ID NO: 2. For example, the invention providesisolated nucleic acid molecules that contain a nucleic acid sequencethat encodes a 15 amino acid sequence identical to any 15 amino acidsequence set forth in SEQ ID NO: 2 including, without limitation, thesequence starting at amino acid residue number 1 and ending at aminoacid residue number 15, the sequence starting at amino acid residuenumber 2 and ending at amino acid residue number 16, the sequencestarting at amino acid residue number 3 and ending at amino acid residuenumber 17, and so forth. Additional examples include, withoutlimitation, isolated nucleic acid molecules that contain a nucleic acidsequence that encodes an amino acid sequence that is 20 or more aminoacid residues (e.g., 21, 22, 23, 24, 25, or more amino acid residues) inlength and identical to any portion of the sequence set forth in SEQ IDNO: 2.

[0059] In addition, the invention provides isolated nucleic acidmolecules that contain a nucleic acid sequence that encodes an aminoacid sequence having a variation of the amino acid sequence set forth inSEQ ID NO: 2. For example, the invention provides isolated nucleic acidmolecules containing a nucleic acid sequence encoding an amino acidsequence set forth in SEQ ID NO: 2 that contains a single insertion, asingle deletion, a single substitution, multiple insertions, multipledeletions, multiple substitutions, or any combination thereof (e.g.,single deletion together with multiple insertions). The invention alsoprovides isolated nucleic acid molecules containing a nucleic acidsequence encoding an amino acid sequence that contains a variant of aportion of the amino acid sequence set forth in SEQ ID NO: 2 asdescribed herein.

[0060] The isolated nucleic acid molecules within the scope of theinvention can encode a polypeptide having mu3 opiate receptor activity.Any method can be use to determine whether or not a particular nucleicacid molecule encodes a polypeptide having mu3 opiate receptor activity.For example, cells transfected with a particular nucleic acid moleculecan be analyzed to determine the expressed polypeptide's bindingaffinity for morphine and DAMGO. If the binding affinity for morphine ishigher than the binding affinity for DAMGO, then the expressedpolypeptide has mu3 opiate receptor activity. Controls can be used toconfirm the specificity of the various binding affinities. For example,untranfected cells can be used to confirm that the measured bindingaffinity is specific for the polypeptide encoded by the introducednucleic acid molecule. Examples of techniques that can be used toevaluate mu3 opiate receptor activities are provided elsewhere (e.g.,WO99/24471).

[0061] Polypeptides

[0062] The invention provides substantially pure polypeptides. The term“substantially pure” as used herein with reference to a polypeptidemeans the polypeptide is substantially free of other polypeptides,lipids, carbohydrates, and nucleic acid with which it is naturallyassociated. Thus, a substantially pure polypeptide is any polypeptidethat is removed from its natural environment and is at least 60 percentpure. A substantially pure polypeptide can be at least about 65, 70, 75,80, 85, 90, 95, or 99 percent pure. Typically, a substantially purepolypeptide will yield a single major band on a non-reducingpolyacrylamide gel.

[0063] Any substantially pure polypeptide having an amino acid sequenceencoded by a nucleic acid within the scope of the invention is itselfwithin the scope of the invention. In addition, any substantially purepolypeptide containing an amino acid sequence having a length and apercent identity to the sequence set forth in SEQ ID NO: 2 over thatlength as determined herein is within the scope of the inventionprovided the point defined by that length and percent identity is withinthe area defined by points A, B, C, and D of FIG. 1; where point A hasan X coordinate less than or equal to 26, and a Y coordinate less thanor equal to 100; where point B has an X coordinate less than or equal to26, and a Y coordinate greater than or equal to 50; where point C has anX coordinate greater than or equal to 10, and a Y coordinate greaterthan or equal to 50; and where point D has an X coordinate greater thanor equal to 5, and a Y coordinate less than or equal to 100. Forexample, the X coordinate for point A can be 26, 25, 24, 23, 22, 21, 20,or less; and the Y coordinate for point A can be 100, 99, 95, 90, 85,80, 75, or less. The X coordinate for point B can be 26, 25, 24, 23, 22,21, 20, or less; and the Y coordinate for point B can be 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 99 or more. The X coordinate for point C can be10, 12, 14, 16, 17, 18, 20, or more; and the Y coordinate for point Ccan be 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or more. The Xcoordinate for point D can be 5, 6, 7, 8, 9, 10, 15, 20, or more; andthe Y coordinate for point D can be 100, 99, 95, 90, 85, 80, 75, orless. In one embodiment, point A can be (26, 100), point B can be (26,95), point C can be (10, 95), and point D can be (5, 100).

[0064] Any method can be used to obtain a substantially purepolypeptide. For example, common polypeptide purification techniquessuch as affinity chromotography and HPLC as well as polypeptidesynthesis techniques can be used. In addition, any material can be usedas a source to obtain a substantially pure polypeptide. For example,tissue from wild-type or transgenic animals can be used as a sourcematerial. In addition, tissue culture cells engineered to over-express aparticular polypeptide of interest can be used to obtain substantiallypure polypeptide. Further, a polypeptide within the scope of theinvention can be engineered to contain an amino acid sequence thatallows the polypeptide to be captured onto an affinity matrix. Forexample, a tag such as c-myc, hemagglutinin, polyhistidine, or Flag™ tag(Kodak) can be used to aid polypeptide purification. Such tags can beinserted anywhere within the polypeptide including at either thecarboxyl or amino termini. Other fusions that could be useful includeenzymes that aid in the detection of the polypeptide, such as alkalinephosphatase.

[0065] The invention provides polypeptides that contain the entire aminoacid sequence set forth in SEQ ID NO: 2, 5, 7, or 9. In addition, theinvention provides polypeptides that contain a portion of the amino acidsequence set forth in SEQ ID NO: 2. For example, the invention providespolypeptides that contain a 5 amino acid sequence identical to any 5amino acid sequence set forth in SEQ ID NO: 2 including, withoutlimitation, the sequence starting at amino acid residue number 1 andending at amino acid residue number 5, the sequence starting at aminoacid residue number 2 and ending at amino acid residue number 6, thesequence starting at amino acid residue number 3 and ending at aminoacid residue number 7, and so forth. It will be appreciated that theinvention also provides polypeptides that contain an amino acid sequencethat is greater than 5 amino acid residues (e.g., 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more aminoacid residues) in length and identical to any portion of the sequenceset forth in SEQ ID NO: 2. For example, the invention providespolypeptides that contain a 15 amino acid sequence identical to any 15amino acid sequence set forth in SEQ ID NO: 2 including, withoutlimitation, the sequence starting at amino acid residue number 1 andending at amino acid residue number 15, the sequence starting at aminoacid residue number 2 and ending at amino acid residue number 16, thesequence starting at amino acid residue number 3 and ending at aminoacid residue number 17, and so forth. Additional examples include,without limitation, polypeptides that contain an amino acid sequencethat is 20 or more amino acid residues (e.g., 21, 22, 23, 24, 25, ormore amino acid residues) in length and identical to any portion of thesequence set forth in SEQ ID NO: 2.

[0066] In addition, the invention provides polypeptides containing anamino acid sequence having a variation of the amino acid sequence setforth in SEQ ID NO: 2. For example, the invention provides polypeptidescontaining an amino acid sequence set forth in SEQ ID NO: 2 thatcontains a single insertion, a single deletion, a single substitution,multiple insertions, multiple deletions, multiple substitutions, or anycombination thereof (e.g., single deletion together with multipleinsertions). The invention also provides polypeptides containing anamino acid sequence that contains a variant of a portion of the aminoacid sequence set forth in SEQ ID NO: 2 as described herein.

[0067] The substantially pure polypeptides within the scope of theinvention can have mu3 opiate receptor activity. Any method can be useto determine whether or not a particular polypeptide has mu3 opiatereceptor activity. For example, cells expressing a particularpolypeptide can be analyzed to determine the polypeptide's bindingaffinity for morphine and DAMGO. If the binding affinity for morphine ishigher than the binding affinity for DAMGO, then the expressedpolypeptide has mu3 opiate receptor activity. Controls can be used toconfirm the specificity of the various binding affinities. For example,cells lacking the polypeptide can be used to confirm that the measuredbinding affinity is specific for that particular polypeptide.

[0068] Host Cells

[0069] A host cell within the scope of the invention is any cellcontaining at least one isolated nucleic acid molecule described herein.Such cells can be prokaryotic and eukaryotic cells. It is noted thatcells containing an isolated nucleic acid molecule within the scope ofthe invention are not required to express a polypeptide. In addition,the isolated nucleic acid molecule can be integrated into the genome ofthe cell or maintained in an episomal state. Thus, host cells can bestably or transiently transfected with a construct containing anisolated nucleic acid molecule of the invention.

[0070] Host cells within the scope of the invention can contain anexogenous nucleic acid molecule that encodes a polypeptide having mu3opiate receptor activity. Such host cells can express the encodedpolypeptide such that the host cells exhibit at least one mu3 opiatereceptor-mediated response after treatment with a mu3 opiate receptoragonist.

[0071] Any methods can be used to introduce an isolated nucleic acidmolecule into a cell in vivo or in vitro. For example, calcium phosphateprecipitation, electroporation, heat shock, lipofection, microinjection,and viral-mediated nucleic acid transfer are common methods that can beused to introduce an isolated nucleic acid molecule into a cell. Inaddition, naked DNA can be delivered directly to cells in vivo asdescribe elsewhere (U.S. Pat. No. 5,580,859 and U.S. Pat. No. 5,589,466including continuations thereof). Further, isolated nucleic acidmolecules can be introduced into cells by generating transgenic animals.

[0072] Transgenic animals can be aquatic animals (such as fish, sharks,dolphin, and the like), farm animals (such as pigs, goats, sheep, cows,horses, rabbits, and the like), rodents (such as rats, guinea pigs, andmice), non-human primates (such as baboon, monkeys, and chimpanzees),and domestic animals (such as dogs and cats). Several techniques knownin the art can be used to introduce isolated nucleic acid molecules intoanimals to produce the founder lines of transgenic animals. Suchtechniques include, without limitation, pronuclear microinjection (U.S.Pat. No. 4,873,191); retrovirus mediated gene transfer into germ lines(Van der Putten et al., Proc. Natl. Acad. Sci., USA, 82:6148(1985));gene transfection into embryonic stem cells (Gossler A et al., Proc NatlAcad Sci USA 83:9065-9069(1986)); gene targeting into embryonic stemcells (Thompson et al., Cell, 56:313(1989)); nuclear transfer of somaticnuclei (Schnieke A E et al., Science 278:2130-2133(1997)); andelectroporation of embryos (Lo C W, Mol. Cell. Biol, 3:1803-1814(1983)).Once obtained, transgenic animals can be replicated using traditionalbreeding or animal cloning.

[0073] Any methods can be used to identify cells containing an isolatednucleic acid molecule of the invention. Such methods include, withoutlimitation, PCR and nucleic acid hybridization techniques such asNorthern and Southern analysis. In some cases, immunohistochemistry andbiochemical techniques can be used to determine if a cell contains aparticular isolated nucleic acid molecule by detecting the expression ofa polypeptide encoded by that particular nucleic acid molecule.

[0074] Identifying Mu3 Opiate Receptor Agonists

[0075] A mu3 opiate receptor agonist is any molecule that interacts witha polypeptide having mu3 opiate receptor activity such that a mu3 opiatereceptor-mediated response is induced. Mu3 opiate receptor-mediatedresponses include, without limitation, changes in intracellular calciumconcentration and nitric oxide release.

[0076] Mu3 opiate receptor agonists can be identified by (1) contactingcells expressing a polypeptide having mu3 opiate receptor activity witha test molecule, and (2) determining if that test molecule induces a mu3opiate receptor-mediated response. Such cells include cells expressing apolypeptide having mu3 opiate receptor activity (e.g., heart cells, veincells, artery cells, testicular cells, and white blood cells) as well ascells containing an isolated nucleic acid molecule that expresses apolypeptide having mu3 opiate receptor activity. For example, a mu3opiate receptor agonist can be identified by contacting cells containingan isolated nucleic acid molecule having a sequence as set forth in SEQID NO: 4, 6, 8, or 10 with a test molecule, and determining if that testmolecule induces changes in intracellular calcium concentration in amu3-specific manner. The specificity of the interaction between apotential mu3 opiate receptor agonist and a mu3 opiate receptor can bedetermined using a known mu3 opiate receptor antagonist. For example, atest molecule that induces a change in intracellular calciumconcentration can be identified as a mu3 opiate receptor agonist if amu3 opiate receptor antagonist can inhibit the induction of that changein intracellular calcium concentration. In addition, the specificity ofagonist-receptor interactions can be demonstrated using heterologousexpression systems, receptor binding analyses, or any other method thatprovides a measure of agonist-receptor interaction.

[0077] A test molecule can be any molecule having any chemicalstructure. For example, a test molecule can be a polypeptide,carbohydrate, lipid, amino acid, nucleic acid, fatty acid, or steroid.In addition, a test molecule can be lipophilic, hydrophilic, plasmamembrane permeable, or plasma membrane impermeable.

[0078] The invention provides several assays that can be used toidentify a mu3 opiate receptor agonist. Such assays involve monitoringat least one of the biological responses mediated by a mu3 opiatereceptor in cells expressing a polypeptide having mu3 opiate receptoractivity such as cells containing an exogenous nucleic acid moleculethat expresses a polypeptide having mu3 opiate receptor activity. Asdescribed herein, mu3 opiate receptor-mediated responses include,without limitation, increases in intracellular calcium concentration andnitric oxide release. Thus, a mu3 opiate receptor agonist can beidentified using an assay that monitors intracellular calciumconcentration, nitric oxide release, or both in cells transfected with anucleic acid molecule that expresses a polypeptide having mu3 opiatereceptor activity.

[0079] Intracellular calcium concentrations can be monitored using anymethod. For example, intracellular calcium concentrations can bemonitored using a dye that detects calcium ions. In this case, cells canbe loaded with a fluorescent dye (e.g., fura-2) and monitored by dualemission microfluorimetry. The fura-2 loading process can involvewashing the cells (e.g., one to four times) with incubation mediumlacking calcium. This medium can be balanced with sucrose to maintainosmolarity. After washing, the cells can be incubated (e.g., 30 minutes)with loading solution. This loading solution can contain, for example, 5μM fura-2/AM and a non-ionic/non-denaturing detergent such as PluronicF-127. The non-ionic/non-denaturing detergent can help disperse theacetoxymethyl (AM) esters of fura-2. After incubation with the loadingsolution, the cells can be washed (e.g., one to four times) with, forexample, PBS without calcium or magnesium to remove extracellular dye.

[0080] Once loaded, the intracellular calcium concentration ([Ca²⁺]i)can be calculated from the fluorescence ratio (340 and 380 nm excitationand 510 nm emission wavelength) according to the following equation:[Ca²⁺]i=(R−R_(min)) k_(d)β/(R_(max)−R); where R=fluorescence ratiorecorded from the cell; R_(min)=fluorescence ratio of fura-2 free acidrecorded in absence of Ca²⁺; R_(max)=fluorescence ratio of fura-2 freeacid recorded in saturating concentration of Ca²⁺; k_(d)=calciumdissociation constant of the dye; and β=the ratio of fluorescence offura-2 free acid in the Ca²⁺ free form to the Ca²⁺ saturated formrecorded at the wavelength used in the denominator of the ratio. Usingan image processing system such as a COMPIX C-640 SIMCA (Compix Inc.,Mars, Pa.) system with an inverted microscope, images can be acquiredfor analysis every 0.4 seconds.

[0081] Nitric oxide (NO) release can be monitored directly or indirectlyusing any method. For example, a NO-specific amperometric probe can beused to measure directly the NO released from cultured cells or tissuefragments as described elsewhere (Stefano G B et al., J. Biol. Chem.270:30290(1995) and Magazine H L et al., J. Immunol. 156:4845(1996)).Using this NO-specific probe, the concentration of NO gas in solutioncan be measured in real-time with, for example, a DUO 18 computer dataacquisition system obtained from World Precision Instruments. Briefly,the cells or tissue fragments can be placed in a superfusion chambercontaining, for example, 2 mL PBS. In addition, a micromanipulator(e.g., a micromanipulator obtained from Zeiss-Eppendorff) can beattached to the stage of an inverted microscope to aid in positioningthe amperometric probe 15 μm above the surface of a cell or tissuefragment. Prior to obtaining measurements, the amperometric probe can becalibrated by generating a standard curve using different concentrationsof a nitrosothiol donor such as S-nitroso-N-acetyl-DL-penicillamine(SNAP) obtained from Sigma (St. Louis, Mo.). In addition, theamperometric probe can be equilibrated in the same solution (e.g., PBS)used to incubate the cells or tissue fragments during analysis.

[0082] Identifying Mu3 Opiate Receptor Antagonists

[0083] A mu3 opiate receptor antagonist is any molecule that interactswith a polypeptide having mu3 opiate receptor activity such that theinduction of a mu3 opiate receptor-mediated response is inhibited orprevented. A mu3 opiate receptor antagonist can be identified by (1)contacting cells expressing a polypeptide having mu3 opiate receptoractivity with a mu3 opiate receptor agonist and a test molecule, and (2)determining if that test molecule inhibits the mu3 opiate receptoragonist from inducing a mu3 opiate receptor-mediated response. Suchcells include cells expressing a polypeptide having mu3 opiate receptoractivity (e.g., heart cells, vein cells, artery cells, testicular cells,and white blood cells) as well as cells containing an isolated nucleicacid molecule that expresses a polypeptide having mu3 opiate receptoractivity. For example, a mu3 opiate receptor antagonist can beidentified by (1) contacting cells transfected with a nucleic acidmolecule that expresses a polypeptide having mu3 opiate receptoractivity with morphine and a test molecule, and (2) determining if thattest molecule inhibits morphine from inducing nitric oxide release.Again, a test molecule can be any molecule having any chemicalstructure. For example, a test molecule can be a polypeptide,carbohydrate, lipid, amino acid, nucleic acid, fatty acid, or steroid.In addition, a test molecule can be lipophilic, hydrophilic, plasmamembrane permeable, or plasma membrane impermeable. The cells can becontacted with the test molecule and the mu3 opiate receptor agonist inany order. For example, the test molecule can be added before the mu3opiate receptor agonist, the test molecule can be added after the mu3opiate receptor agonist, or the test molecule and mu3 opiate receptoragonist can be added simultaneously.

[0084] It is to be understood that each of the assays for identifyingmu3 opiate receptor agonists described herein can be adapted such thatmu3 opiate receptor antagonists can be identified.

[0085] The invention will be further described in the followingexamples, which do not limit the scope of the invention described in theclaims.

EXAMPLES Example 1 Nucleic Acid Encoding a Polypeptide Having Mu3 OpiateReceptor Activity

[0086] A human testis cDNA library constructed in the pEXP1 mammalianexpression vector was obtained from Clonetech (Palo Alto, Calif.). Afterobtaining a DNA sample from the human testis cDNA library, the librarywas prescreened by PCR using primers designed to amplify a 441 base pairregion of the human mul opioid receptor. The forward primer had asequence corresponding to position 892-919 of the human mu1 opioidreceptor (5′-GGTACTGGGAAAACCTGCTGAAGATCTG-3′; SEQ ID NO: 14), and thereverse primer had a sequence corresponding to position 1305-1332 of thehuman mu1 opioid receptor (5′-GGTCTCTAGTGTTCTGACGAATTCGAGT-3′; SEQ IDNO: 15). After the amplification reaction, the amplification productswere separated by gel electrophoresis using a 2% agarose gel stainedwith ethidium bromide. A 441 base pair fragment was observed.

[0087] The human testis cDNA library was screened with a probe madeusing the same forward and reverse primers. Briefly, the screen wasperformed using ClonCapture cDNA Selection Kit (CloneTech; Palo Alto,Calif.) according to the manufacturer's instructions. Two positivecolonies were identified in the enrichment library screen. PCR confirmedthat the two colonies were positive. After isolation, the plasmid DNAfrom the two colonies was digested with SfiI and separated by gelelectrophoresis. One insert was found to be about 1.1 kb in size whilethe other was found to be about 2.0 kb in size. Each insert wassequenced.

[0088] Sequence analysis of the 2.0 kb insert revealed a new splicevariant that replaces the last 12 amino acid residues of the human mulopioid receptor with 26 different amino acid residues. Specifically, thenucleic acid sequence that encodes the LENLEAETAPLP (SEQ ID NO: 16)carboxyl-terminus sequence of the mu1 opioid receptor was found to bereplaced with a nucleic acid sequence that encodesNYYIIHRLCCNTPLISQKPV-LLWFCD (SEQ ID NO: 2). The nucleic acid sequenceencoding NYYIIHRLCC-NTPLISQKPVLLWFCD (SEQ ID NO: 2) was found to be5′-AATTATTATATAAT-TCATAGATGTTGCTGCAATACCCCTCTTATTTCTCAAAAGCCAGTCTTGCTCTGGTTCTGTGATTAA-3′ (SEQ ID NO: 1). The following nucleic acid sequencecontains the sequence set forth in SEQ ID NO: 1 as well as the sequencefound to extend past the TAA stop codon:5′-AATTATTATATAATTCATAGATGTTGCTGC-AATACCCCTCTTATTTCTCAAAAGCCAGTCTTGCTCTGGTTCTGTGATTAAAGAGAGAGGGTGAGTGCCTTGCCCACTGTGGTCATGGATGCAAGATATTCACAGAAAATTAGCATCATAGAAAAAAAANNNAAAAAAAAAAAAAAAAAANCATGTCGGCCGCCTCGGCCAAACATCGGGTCGAGCATGCATCTAGGGCGGCCAATTCCGCCCCTCTCCCCCCCNGCNNTTT (SEQ ID NO: 3). This sequence was found toreplace the sequence of SEQ ID NO: 12 that corresponds to nucleotidenumber 1374-1826 as follows:5′-CTAGAAAATCTGGAAGCAGAAACTGCTCCGTTGCCCTAACAGG-GTCTCATGCCATTCCGACCTTCACCAAGCTTAGAAGCCACCATGTATGTGGAAGCAGGTTGCTTCAAGAATGTGTAGGAGGCTCTAATTCTCTAGGAAAGTGCCTGCTTTTAGGTCATCCAACCTCTTTCCTCTCTGGCCACTCTGCTCTGCACATTAGAGGGACAGCCAAAAGTAAGTGGAGCATTTGGAAGGAAAGGAATATACCACACCGAGGAGTCCAGTTTGTGCAAGACACCCAGTGGAACCAAAACCCATCGTGGTATGTGAATTGAAGTCATCATAAAAGGTGACCCTTCTGTCTGTAAGATTTTATTTTCAAGCAAATATTTATGACCTCAACAAAGAAGAACCATCTTTTGTTAAGTTCACCGTAGTAACACATAAAGTAAATGCTACCTCTGATCAAAG-3′ (SEQ ID NO: 18).

[0089] The following nucleic acid sequence encodes a polypeptide thatuses the start codon of the human mul opioid receptor and thecarboxyl-terminus of the 2.0 kb insert:5′-ATGTCAGATGCTCAGCTCGGTCCCCTCCGCCTGACGCTCCTCTCTGTCT-CAGCCAGGACTGGTTTCTGTAAGAAACAGCAGGAGCTGTGGCAGCGGCGAAAGGAAGCGGCTGAGGCGCTTGGAACCCGAAAAGTCTCGGTGCTCCTGGCTACCTCGCACAGCGGTGCCCGCCCGGCCGTCAGTACCATGGACAGCAGCGCTGCCCCCACGAACGCCAGCAATTGCACTGATGCCTTGGCGTACTCAAGTTGCTCCCCAGCACCCAGCCCCGGTTCCTGGGTCAACTTGTCCCACTTAGATGGCAACCTGTCCGACCCATGCGGTCCGAACCGCACCGACCTGGGCGGGAGAGACAGCCTGTGCCCTCCGACCGGCAGTCCCTCCATGATCACGGCCATCACGATCATGGCCCTCTACTCCATCGTGTGCGTGGTGGGGCTCTTCGGAAACTTCCTGGTCATGTATGTGATTGTCAGATACACCAAGATGAAGACTGCCACCAACATCTACATTTTCAACCTTGCTCTGGCAGATGCCTTAGCCACCAGTACCCTGCCCTTCCAGAGTGTGAATTACCTAATGGGAACATGGCCATTTGGAACCATCCTTTGCAAGATAGTGATCTCCATAGATTACTATAACATGTTCACCAGCATATTCACCCTCTGCACCATGAGTGTTGATCGATACATTGCAGTCTGCCACCCTGTCAAGGCCTTAGATTTCCGTACTCCCCGAAATGCCAAAATTATCAATGTCTGCAACTGGATCCTCTCTTCAGCCATTGGTCTTCCTGTAATGTTCATGGCTACAACAAAATACAGGCAAGGTTCCATAGATTGTACACTAACATTCTCTCATCCAACCTGGTACTGGGAAAACCTGCTGAAGATCTGTGTTTTCATCTTCGCCTTCATTATGCCAGTGCTCATCATTACCGTGTGCTATGGACTGATGATCTTGCGCCTCAAGAGTGTCCGCATGCTCTCTGGCTCCAAAGAAAAGGACAGGAATCTTCGAAGGATCACCAGGATGGTGCTGGTGGTGGTGGCTGTGTTCATCGTCTGCTGGACTCCCATTCACATTTACGTCATCATTAAAGCCTTGGTTACAATCCCAGAAACTACGTTCCAGACTGTTTCTTGGCACTTCTGCATTGCTCTAGGTTACACAAACAGCTGCCTCAACCCAGTCCTTTATGCATTTCTGGATGAAAACTTCAAACGATGCTTCAGAGAGTTCTGTATCCCAACCTCTTCCAACATTGAGCAACAAAACTCCACTCGAATTCGTCAGAACACTAGAGACCACCCCTCCACGGCCAATACAGTGGATAGAACTAATCATCAGAATTATTATATAATTCATAGATGTTGCTGCAATACCCCTCTTATTTCTCAAAAGCCAGTCTTGCTCTGG TTCTGTGATTAA-3′(SEQ ID NO: 6). The amino acid sequence encoded by this nucleic acidsequence is as follows:MSDAQLGPLRLTLLSVSARTGFCKKQQEL-WQRRKEAAEALGTRKVSVLLATSHSGARPAVSTMDSSAAPTNASNCTDALAYSSCSPAPSPGSWVNLSHLDGNLSDPCGPNRTDLGGRDSLCPPTGSPSMITAITIMALYSIVCVVGLFGNFLVMYVIVRYTKMKTATNIYIFNLALADALATSTLPFQSVNYLMGTWPFGTILCKIVISIDYYNMFTSIFTLCTMSVDRYIAVCHPVKALDFRTPRNAKIINVCNWILSSAIGLPVMFMATTKYRQGSIDCTLTFSHPTWYWENLLKICVFIFAFIMPVLIITVCYGLMILRLKSVRMLS GSKEKDRNLRRITRMVLVVVAVFIVCWTPIHIYVIIKALVTIPETTFQTVSWHFCIALGYTNSCLNPVLYAFLDENFKRCFREFCIPTSSNIEQQNSTRIRQNTRDHPSTANTVDRTNHQNYYIIHRLCCNTPLISQKPVLLWFC D (SEQ ID NO:7).

[0090] The following nucleic acid sequence encodes a polypeptide thatuses the start codon of the human mu2 opioid receptor and thecarboxyl-terminus of the 2.0 kb insert:5′-ATGGACAGCAGCGCTGCCCCCACGAACGCCAGCAATTGCACTGATGC-CTTGGCGTACTCAAGTTGCTCCCCAGCACCCAGCCCCGGTTCCTGGGTCAACTTGTCCCACTTAGATGGCAACCTGTCCGACCCATGCGGTCCGAACCGCACCGACCTGGGCGGGAGAGACAGCCTGTGCCCTCCGACCGGCAGTCCCTCCATGATCACGGCCATCACGATCATGGCCCTCTACTCCATCGTGTGCGTGGTGGGGCTCTTCGGAAACTTCCTGGTCATGTATGTGATTGTCAGATACACCAAGATGAAGACTGCCACCAACATCTACATTTTCAACCTTGCTCTGGCAGATGCCTTAGCCACCAGTACCCTGCCCTTCCAGAGTGTGAATTACCTAATGGGAACATGGCCATTTGGAACCATCCTTTGCAAGATAGTGATCTCCATAGATTACTATAACATGTTCACCAGCATATTCACCCTCTGCACCATGAGTGTTGATCGATACATTGCAGTCTGCCACCCTGTCAAGGCCTTAGATTTCCGTACTCCCCGAAATGCCAAAATTATCAATGTCTGCAACTGGATCCTCTCTTCAGCCATTGGTCTTCCTGTAATGTTCATGGCTACAACAAAATACAGGCAAGGTTCCATAGATTGTACACTAACATTCTCTCATCCAACCTGGTACTGGGAAAACCTGCTGAAGATCTGTGTTTTCATCTTCGCCTTCATTATGCCAGTGCTCATCATTACCGTGTGCTATGGACTGATGATCTTGCGCCTCAAGAGTGTCCGCATGCTCTCTGGCTCCAAAGAAAAGGACAGGAATCTTCGAAGGATCACCAGGATGGTGCTGGTGGTGGTGGCTGTGTTCATCGTCTGCTGGACTCCCATTCACATTTACGTCATCATTAAAGCCTTGGTTACAATCCCAGAAACTACGTTCCAGACTGTTTCTTGGCACTTCTGCATTGCTCTAGGTTACACAAACAGCTGCCTCAACCCAGTCCTTTATGCATTTCTGGATGAAAACTTCAAACGATGCTTCAGAGAGTTCTGTATCCCAACCTCTTCCAACATTGAGCAACAAAACTCCACTCGAATTCGTCAGAACACTAGAGACCACCCCTCCACGGCCAATACAGTGGATAGAACTAATCATCAGAATTATTATATAATTCATAGATGTTGCTGCAATACCCCTCTTATTTCTCAAAAGCCAGTCTTGCTCTGGTTCTGTGATTAA-3′ (SEQ ID NO: 8). The aminoacid sequence encoded by this nucleic acid sequence is as follows:MDS-SAAPTNASNCTDALAYSSCSPAPSPGSWVNLSHLDGNLSDPCGPNRTDLGGRDSLCPPTGSPSMITAITIMALYSIVCVVGLFGNFLVMYVIVRYTKMKTATNIYIFNLALADALATSTLPFQSVNYLMGTWPFGTILCKIVISIDYYNMFTSIFTLCTMSVDRYIAVCHPVKALDFRTPRNAKIINVCNWILSSAIGLPVMFMATTKYRQGSIDCTLTFSHPTWYWENLLKICVFIFAFIMPVLIITVCYGLMILRLKSVRMLSGSKEKDRNLRRITRMVLVVVAVFIVCWTPIHIYVIIKALVTIPETTFQTVSWHFCIALGYTNSCLNPVLYAFLDENFKRCFREFCIPTSSNIEQQNSTRIRQNTRDHPSTANTVDRTNHQNYYIIHRLCCNTPLISQKPVLLWFCD (SEQ ID NO: 9).

[0091] The following nucleic acid sequence encodes a polypeptide thatuses the start codon of the rat mu2 opioid receptor and thecarboxyl-terminus of the 2.0 kb insert:5′-ATGGACAGCAGCACCGGCCCAGGGAACACCAGCGACTGCTCAGACCCCTTAGCTCAGGCAAGTTGCTCCCCAGCACCTGGCTCCTGGGTCAACTTGTCCCACTTAGATGGCAACCTGTCCGACCCATGCGGTCCGAACCGCACCGACCTGGGCGGGAGAGACAGCCTGTGCCCTCCGACCGGCAGTCCCTCCATGATCACGGCCATCACGATCATGGCCCTCTACTCCATCGTGTGCGTGGTGGGGCTCTTCGGAAACTTCCTGGTCATGTATGTGATTGTCAGATACACCAAGATGAAGACTGCCACCAACATCTACATTTTCAACCTTGCTCTGGCAGATGCCTTAGCCACCAGTACCCTGCCCTTCCAGAGTGTGAATTACCTAATGGGAACATGGCCATTTGGAACCATCCTTTGCAAGATAGTGATCTCCATAGATTACTATAACATGTTCACCAGCATATTCACCCTCTGCACCATGAGTGTTGATCGATACATTGCAGTCTGCCACCCTGTCAAGGCCTTAGATTTCCGTACTCCCCGAAATGCCAAAATTATCAATGTCTGCAACTGGATCCTCTCTTCAGCCATTGGTCTTCCTGTAATGTTCATGGCTACAACAAAATACAGGCAAGGTTCCATAGATTGTACACTAACATTCTCTCATCCAACCTGGTACTGGGAAAACCTGCTGAAGATCTGTGTTTTCATCTTCGCCTTCATTATGCCAGTGCTCATCATTACCGTGTGCTATGGACTGATGATCTTGCGCCTCAAGAGTGTCCGCATGCTCTCTGGCTCCAAAGAAAAGGACAGGAATCTTCGAAGGATCACCAGGATGGTGCTGGTGGTGGTGGCTGTGTTCATCGTCTGCTGGACTCCCATTCACATTTACGTCATCATTAAAGCCTTGGTTACAATCCCAGAAACTACGTTCCAGACTGTTTCTTGGCACTTCTGCATTGCTCTAGGTTACACAAACAGCTGCCTCAACCCAGTCCTTTATGCATTTCTGGATGAAAACTTCAAACGATGCTTCAGAGAGTTCTGTATCCCAACCTCTTCCAACATTGAGCAACAAAACTCCACTCGAATTCGTCAGAACACTAGAGACCACCCCTCCACGGCCAATACAGTGGATAGAACTAATCATCAGAATTATTATATAATTCATAGATGTTGCTGCAATACCCCTCTTATTTCTCAAAAGCCAGTCTTGCTCTGGTTCTGTGATTAA-3′ (SEQ ID NO: 10). The amino acid sequenceencoded by this nucleic acid sequence is as follows:MDSSTGPGNTSD-CSDPLAQASCSPAPGSWVNLSHLDGNLSDPCGPNRTDLGGRDSLCPPTGSPSMITAITIMALYSIVCVVGLFGNFLVMYVIVRYTKMKTATNIYIFNLALADALATSTLPFQSVNYLMGTWPFGTILCKIVISIDYYNMFTSIFTLCTMSVDRYIAVCHPVKALDFRTPRNAKIINVCNWILSSAIGLPVMFMATTKYRQGSIDCTLTFSHPTWYWENLLKICVFIFAFIMPVLIITVCYGLMILRLKSVRMLSGSKEKDRNLRRITRMVLVVVAVFIVCWTPIHIYVIIKALVTIPETTFQTVSWHFCIALGYTNSCLNPVLYAFLDENFKRCFR EFCIPTSSNIEQQNSTRIRQNTRDHPSTANTVDRTNHQNYYIIHRLCCNTPLISQKP VLLWFCD (SEQ ID NO:11).

[0092] In addition, sequence analysis revealed that the 2.0 kb insertlacked the first exon of the human mu1 opioid receptor. Specifically,the 5′ end of the 2.0 kb insert started with 5′-ATACACCAAGATG-3′ (SEQ IDNO: 17), lacking the first 498 nucleotides of the mu1 opioid receptornucleic acid sequence reported in GenBank® accession numberXM_(—)004341, which is set forth in SEQ ID NO: 12. The following nucleicacid sequence corresponds to the open reading frame of the 2.0 kbinsert: 5′-ATGAAGACTGCCA-CCAACATCTACATTTTCAACCTTGCTCTGGCAGATGCCTTAGCCACCAGTACCCTGCCCTTCCAGAGTGTGAATTACCTAATGGGAACATGGCCATTTGGAACCATCCTTTGCAAGATAGTGATCTCCATAGATTACTATAACATGTTCACCAGCATATTCACCCTCTGCACCATGAGTGTTGATCGATACATTGCAGTCTGCCACCCTGTCAAGGCCTTAGATTTCCGTACTCCCCGAAATGCCAAAATTATCAATGTCTGCAACTGGATCCTCTCTTCAGCCATTGGTCTTCCTGTAATGTTCATGGCTACAACAAAATACAGGCAAGGTTCCATAGATTGTACACTAACATTCTCTCATCCAACCTGGTACTGGGAAAACCTGCTGAAGATCTGTGTTTTCATCTTCGCCTTCATTATGCCAGTGCTCATCATTACCGTGTGCTATGGACTGATGATCTTGCGCCTCAAGAGTGTCCGCATGCTCTCTGGCTCCAAAGAAAAGGACAGGAATCTTCGAAGGATCACCAGGATGGTGCTGGTGGTGGTGGCTGTGTTCATCGTCTGCTGGACTCCCATTCACATTTACGTCATCATTAAAGCCTTGGTTACAATCCCAGAAACTACGTTCCAGACTGTTTCTTGGCACTTCTGCATTGCTCTAGGTTACACAAACAGCTGCCTCAACCCAGTCCTTTATGCATTTCTGGATGAAAACTTCAAACGATGCTTCAGAGAGTTCTGTATCCCAACCTCTTCCAACATTGAGCAACAAAACTCCACTCGAATTCGTCAGAACACTAGAGACCACCCCTCCACGGCCAATACAGTGGATAGAACTAATCATCAGAATTATTATATAATTCATAGATGTTGCTGCAATACCCCTCTTATTTCTCAAAAGCCAGTCTTGCTCTGGTTCTGTGATTAA (SEQ ID NO: 4). The amino acid sequenceencoded by this open reading frame is as follows:MKTATNIYIFNLALAD-ALATSTLPFQSVNYLMGTWPFGTILCKIVISIDYYNMFTSIFTLCTMSVDRYIAVCHPVKALDFRTPRNAKIINVCNWILSSAIGLPVMFMATTKYRQGSIDCTLTFSHPTWYWENLLKICVFIFAFIMPVLIITVCYGLMILRLKSVRMLSGSKEKDRNLRRITRMVLVVVAVFIVCWTPIHIYVIIKALVTIPETTFQTVSWHFCIALGYTNSCLNPVLYAFLDENFKRCFREFCIPTSSNIEQQNSTRIRQNTRDHPSTANTVDRTNHQNYYIIHRLCCNTPLISQKPVLLWFCD (SEQ ID NO: 5).

[0093] The nucleic acid sequence reported for the human mul opioidreceptor is as follows:5′-GAGGGGGCTATACGCAGAGGAGAATGTCAGATGCTCAGCTCGGT-CCCCTCCGCCTGACGCTCCTCTCTGTCTCAGCCAGGACTGGTTTCTGTAAGAAACAGCAGGAGCTGTGGCAGCGGCGAAAGGAAGCGGCTGAGGCGCTTGGAACCCGAAAAGTCTCGGTGCTCCTGGCTACCTCGCACAGCGGTGCCCGCCCGGCCGTCAGTACCATGGACAGCAGCGCTGCCCCCACGAACGCCAGCAATTGCACTGATGCCTTGGCGTACTCAAGTTGCTCCCCAGCACCCAGCCCCGGTTCCTGGGTCAACTTGTCCCACTTAGATGGCAACCTGTCCGACCCATGCGGTCCGAACCGCACCGACCTGGGCGGGAGAGACAGCCTGTGCCCTCCGACCGGCAGTCCCTCCATGATCACGGCCATCACGATCATGGCCCTCTACTCCATCGTGTGCGTGGTGGGGCTCTTCGGAAACTTCCTGGTCATGTATGTGATTGTCAGATACACCAAGATGAAGACTGCCACCAACATCTACATTTTCAACCTTGCTCTGGCAGATGCCTTAGCCACCAGTACCCTGCCCTTCCAGAGTGTGAATTACCTAATGGGAACATGGCCATTTGGAACCATCCTTTGCAAGATAGTGATCTCCATAGATTACTATAACATGTTCACCAGCATATTCACCCTCTGCACCATGAGTGTTGATCGATACATTGCAGTCTGCCACCCTGTCAAGGCCTTAGATTTCCGTACTCCCCGAAATGCCAAAATTATCAATGTCTGCAACTGGATCCTCTCTTCAGCCATTGGTCTTCCTGTAATGTTCATGGCTACAACAAAATACAGGCAAGGTTCCATAGATTGTACACTAACATTCTCTCATCCAACCTGGTACTGGGAAAACCTGCTGAAGATCTGTGTTTTCATCTTCGCCTTCATTATGCCAGTGCTCATCATTACCGTGTGCTATGGACTGATGATCTTGCGCCTCAAGAGTGTCCGCATGCTCTCTGGCTCCAAAGAAAAGGACAGGAATCTTCGAAGGATCACCAGGATGGTGCTGGTGGTGGTGGCTGTGTTCATCGTCTGCTGGACTCCCATTCACATTTACGTCATCATTAAAGCCTTGGTTACAATCCCAGAAACTACGTTCCAGACTGTTTCTTGGCACTTCTGCATTGCTCTAGGTTACACAAACAGCTGCCTCAACCCAGTCCTTTATGCATTTCTGGATGAAAACTTCAAACGATGCTTCAGAGAGTTCTGTATCCCAACCTCTTCCAACATTGAGCAACAAAACTCCACTCGAATTCGTCAGAACACTAGAGACCACCCCTCCACGGCCAATACAGTGGATAGAACTAATCATCAGCTAGAAAATCTGGAAGCAGAAACTGCTCCGTTGCCCTAACAGGGTCTCATGCCATTCCGACCTTCACCAAGCTTAGAAGCCACCATGTATGTGGAAGCAGGTTGCTTCAAGAATGTGTAGGAGGCTCTAATTCTCTAGGAAAGTGCCTGCTTTTAGGTCATCCAACCTCTTTCCTCTCTGGCCACTCTGCTCTGCACATTAGAGGGACAGCCAAAAGTAAGTGGAGCATTTGGAAGGAAAGGAATATACCACACCGAGGAGTCCAGTTTGTGCAAGACACCCAGTGGAACCAAAACCCATCGTGGTATGTGAATTGAAGTCATCATAAAAGGTGACCCTTCTGTCTGTAAGATTTTATTTTCAAGCAAATATTTATGACCTCAACAAAGAAGAACCATCTTTTGTTAAGTTCACCGTAGTAACACATAAAGTAAATGCTACCTCTGATCAAAGCACCTTGAATGGAAGGTCCGAGTCTTTTTAGTGTTTTGCAAGGGAATGAATCCATTATTCTATTTTAGACTTTTAACTTCACCTTAAAATTAGCATCTGGCTAAGGCATCATTTTCACCTCCATTTCTTGGTTTTGTATTGTTTAAAAAAATAACATCTCTTTCATCTAGCTCCATAATTGCAAGGGAAGAGATTAGCATGAAAGGTAATCTGAAACACAGTCATGTGTCAGCTGTAGAAAGGTTGATTCTCATGCACTGCAAATACTTCCAAAGAGTCATCATGGGGGATTTTTCATTCTTAGGCTTTCAGTGGTTTGTTC C-3′ (SEQ IDNO:12).

[0094] The nucleic acid sequence reported for the human mu2 opioidreceptor is as follows:5′-GCAGAGGAGAATGTCAGATGCTCAGCTCGGTCCCCTCCGCCTGA-CGCTCCTCTCTGTCTCAGCCAGGACTGGTTTCTGTAAGAAACAGCAGGAGCTGTGGCAGCGGCGAAAGGAAGCGGCTGAGGCGCTTGGAACCCGAAAAGTCTCGGTGCTCCTGGCTACCTCGCACAGCGGTGCCCGCCCGGCCGTCAGTACCATGGACAGCAGCGCTGCCCCCACGAACGCCAGCAATTGCACTGATGCCTTGGCGTACTCAAGTTGCTCCCCAGCACCCAGCCCCGGTTCCTGGGTCAACTTGTCCCACTTAGATGGCGACCTGTCCGACCCATGCGGTCCGAACCGCACCGACCTGGGCGGGAGAGACAGCCTGTGCCCTCCAACCGGCAGTCCCTCCATGATCACGGCCATCACGATCATGGCCCTCTACTCCATCGTGTGCGTGGTGGGGCTCTTCGGAAACTTCCTGGTCATGTAGTGATTGTCAGATACACCAAGATGAAGACTGCCACCAACATCTACATTTTCAACCTTGCTCTGGCAGATGCCTTAGCCACCAGTACCCTGCCCTTCCAGAGTGTGAATTACCTAATGGGAACATGGCCATTTGGAACCATCCTTTGCAAGATAGTGATCTCCATAGATTACTATAACATGTTCACCAGCATATTCACCCTCTGCACCATGAGTGTTGATCGATACATTGCAGTCTGCCACCCTGTCAAGGCCTTAGATTTCCGFACTCCCCGAAATGCCAAAATTATCAATGTCTGCAACTGGATCCTCTCTTCAGCCATTGGTCTTCCTGTAATGTTCATAGCTACAACAAAATACAGGCAAGGTTCCATAGATTGTACACTAACATTCTCTCATCCAACCTGGTACTGGGAAAACCTGCTGAAGATCTGTGTTTTCATCTTCGCCTTCATTATGCCAGTGCTCATCATTACCGTGTGCTATGGACTGATGATCTTGCGCCTCAAGAGTGTCCGCATGCTCTCTGGCTCCAAAGAAAAGGACAGGAATCTTCGAAGGATCACCAGGATGGTGCTGGTGGTGGTGGCTGTGTTCATCGTCTGCTGGACTCCCATTCACATTTACGTCATCATTAAAGCCTTGGTTACAATCCCAGAAACTACGTTCCAGACTGTTTCTTGGCACTTCTGCATTGCTCTAGGTTACACAAACAGCTGCCTCAACCCAGTCCTTTATGCATTTCTGGATGAAAACTTCAAACGATGCTTCAGAGAGTTCTGTATCCCAACCTCTTCCAACATTGAGCAACAAAACTCCACTCGAATTCGTCAGAACACTAGAGACCACCCCTCCACGGCCAATACAGTGGATAGAACTAATCATCAGGTACGCAGTCTCTAGAATTAGGTATATCTACTGGGGATGACATAAAAATTATAAGGCTTTGTGCTAAACTAGGAGTTTAATCCATTATAGAGGATGAGAATGG AGGGAAGCTT-3′(SEQ.ID NO: 13).

Example 2 Detecting Mu3 Opiate Receptor Expression

[0095] Human heart, vein, and artery tissue samples were homogenized inTRI REAGENT (Molecular Research Center, Inc., Cincinnati, Ohio) using apolytron homogenizer. Human white blood cell samples were homogenized inTRI REAGENT by passing the samples through a 1 mL pipette ten times. Thehomogenates were stored at room temperature for 5 minutes to allowcomplete dissociation of nucleoprotein. 0.1 mL of1-bromo-3-chloropropane (BCP) per 1 mL of TRI Reagent was added to thehomogenates. The samples were vortexed vigorously for 15 seconds andthen stored at room temperature for 7 minutes. After centrifugation ofthe samples for 15 minutes at 12,000 g, the aqueous phase wastransferred to a fresh tube. RNA was precipitated by mixing with 0.5 mLof isopropanol per 1 mL of TRI REAGENT used in for the initialhomogenization. Samples were stored at room temperature for 6 minutesand then centrifuged at 12,000 g for 8 minutes at 4° C. After removingthe supernatant, the RNA pellet was washed with 1 mL of 75% ethanol per1 mL TRI REAGENT used for the initial homogenization, and subsequentlycentrifuged at 7,500 g for 5 minutes at 4° C. The ethanol was discarded,and the RNA pellet air-dried for 5 minutes. The RNA pellet was dissolvedin water and used as template.

[0096] An aliquot of each RNA sample was separated in an 1% agarose gelstained with ethidium bromide. Two predominant bands of small (˜2 kb)and large (˜5 kb) ribosomal RNA were observed. In addition,spectrophotometric measurements of the RNA samples were analyzed at 260and 280 nm. The 260/280 ratios from all of the samples were above 1.6.

[0097] PCR analysis was used to study the expression of mRNA encoding ahuman mu3 opiate receptor. Briefly, PCR analysis was performed using thefollowing primers: 5′-GGTACTGGGAAAACCTGCTGAAGATCTGTG-3′ (SEQ ID NO: 19)and 5′-CATCCATGACCACAGTGGGCAAGGCAC-3′ (SEQ ID NO: 20). Separation of thePCR products by gel electrophoresis revealed a large (about 910 bp) andsmall (about 605 bp) band for each of the four tissue samples (humanheart, vein, and artery tissue and human white blood cells). Theintensity of the large band for the human white blood cell sample wasgreater than the intensity of the large band for the human heart, vein,and artery samples. In addition, the intensity of the small band wasabout the same for the four samples. This result indicates that the mRNAcorresponding to the larger band is expressed at a higher level in whiteblood cells when compared to its level of expression in vascular tissue.

[0098] Each band from each sample was purified, cloned into a TA cloningvector, and sequenced. The smaller band (about 605 bp) had a nucleicacid sequence corresponding to the nucleic acid sequence that encodes ahuman mu3 opiate receptor (e.g., SEQ ID NO: 4). The larger band (about910 bp) had the following nucleic acid sequence:5′-TG-GTGCTGGTGGTGGTGGCTGTGTTCATCGTCTGCTGGACTCCCATTCACATTTACGTCATCATTAAAGCCTTGGTTACAATCCCAGAAACTACGTTCCAGACTGTTTCTTGGCACTTCTGCATTGCTCTAGGTTACACAAACAGCTGCCTCAACCCAGTCCTTTATGCATTTCTGGATGAAAACTTCAAACGATGCTTCAGAGAGTTCTGTATCCCAACCTCTTCCAACATTGAGCAACAAAACTCCACTCGAATTCGTCAGAACACTAGAGACCACCCCTCCACGGCCAATACAGTGGATAGAACTAATCATCAGGTACGCAGTCTCTAGAATTAGGTATATCTACTGGGGATGACATAAAAATTATAAGGCTTTGTGCTAAACTAGGAGTTTAATCCATTATAGAGGATGAGAATGGAGGAAGGGAAAGCAAATTGTGGTTTAAGGGTTAAAGAAGAGGTTTGTATATAAACTGGGGTCCTTTAAATTTGCCTGTACATATTCATTAAGGTTTAAGGATCCCCAATGGGNAAAACCATGGAACTTTTCAAAATACCTTTTTTATGGCCTTTACTTTTATGCAAAATTTATGACTTTAGCACATTATAGAAATAATTCTGATCTAGAATCCTTTTCATTTTCCCCAGAATTATTATATAATTCATAGATGTTCTGCAATACCCCTCTTATTTCTCAAAAGCCAGTCTTGCTCTGGTTTCTGGATTAAAGAGAGAGGGTGAGTGCCTTGCCCACTGTGGTCATGGATGCAAGATATTCACAGAAAATTAGCATCATAGAAAAAAAANNNAAAAAAAAAAAAAAAAAANCATGTCGGCCGCCTCGGCCAAACATCGGGTCGAGCATGCATCTAGGGCGGCCAATTCCGCCCCTCT CCCCCCCNGCNNTTT-3′(SEQ ID NO: 21). The mRNA corresponding to the 910 bp band wasdesignated a mu4 opiate receptor, while the mRNA corresponding to the605 bp band was designated a mu3 opiate receptor.

[0099] Real time RT-PCR was performed using the same primers and RNAsamples. The results confirmed that the mu3 mRNA is expressed equally inhuman heart, vein, artery, and white blood cells. In addition, theresults confirmed that the mu4 mRNA is expressed to a greater extent inhuman heart, vein, and artery than in human white blood cells.

[0100] The following nucleic acid sequence was unique to the mu4 opiatereceptor sequence:5′-GGAAGGGAAAGCAAATTGTGGTTTAAGGGTTAAAGAAGAGGT-TTGTATATAAACTGGGGTCCTTTAAATTTGCCTGTACATATTCATTAAGGTTTAAGGATCCCCAATGGGNAAAACCATGGAACTTTTCAAAATACCTTTTTTATGGCCTTTACTTTTATGCAAAATTTATGACTTTAGCACATTATAGAAATAATTCTGATCTAGAATCCTTTTCATTTTCCC-3′ (SEQ ID NO: 22). The following nucleic acidsequence corresponds to the 5′ end of SEQ ID NO: 4 and the 3′ end of SEQID NO: 21:5′-ATACACCAAGATGAAGACTGCCACCAACATCTACATTTTCAACCT-TGCTCTGGCAGATGCCTTAGCCACCAGTACCCTGCCCTTCCAGAGTGTGAATTACCTAATGGGAACATGGCCATTTGGAACCATCCTTTGCAAGATAGTGATCTCCATAGATTACTATAACATGTTCACCAGCATATTCACCCTCTGCACCATGAGTGTTGATCGATACATTGCAGTCTGCCACCCTGTCAAGGCCTTAGATTTCCGTACTCCCCGAAATGCCAAAATTATCAATGTCTGCAACTGGATCCTCTCTTCAGCCATTGGTCTTCCTGTAATGTTCATAGCTACAACAAAATACAGGCAAGGTTCCATAGATTGTACACTAACATTCTCTCATCCAACCTGGTACTGGGAAAACCTGCTGAAGATCTGTGTTTTCATCTTCGCCTTCATTATGCCAGTGCTCATCATTACCGTGTGCTATGGACTGATGATCTTGCGCCTCAAGAGTGTCCGCATGCTCTCTGGCTCCAAAGAAAAGGACAGGAATCTTCGAAGGATCACCAGGATGGTGCTGGTGGTGGTGGCTGTGTTCATCGTCTGCTGGACTCCCATTCACATTTACGTCATCATTAAAGCCTTGGTTACAATCCCAGAAACTACGTTCCAGACTGTTTCTTGGCACTTCTGCATTGCTCTAGGTTACACAAACAGCTGCCTCAACCCAGTCCTTTATGCATTTCTGGATGAAAACTTCAAACGATGCTTCAGAGAGTTCTGTATCCCAACCTCTTCCAACATTGAGCAACAAAACTCCACTCGAATTCGTCAGAACACTAGAGACCACCCCTCCACGGCCAATACAGTGGATAGAACTAATCATCAGGTACGCAGTCTCTAGAATTAGGTATATCTACTGGGGATGACATAAAAATTATAAGGCTTTGTGCTAAACTAGGAGTTTAATCCATTATAGAGGATGAGAATGGAGGGAAGGGAAAGCAAATTGTGGTTTAAGGGTTAAAGAAGAGGTTTGTATATAAACTGGGGTCCTTTAAATTTGCCTGTACATATTCATTAAGGTTTAAGGATCCCCAATGGGNAAAACCATGGAACTTTTCAAAATACCTTTTTTATGGCCTTTACTTTTATGCAAAATTTATGACTTTAGCACATTATAGAAATAATTCTGATCTAGAATCCTTTTCATTTTCCCCAGAATTATTATATAATTCATAGATGTTCTGCAATACCCCTCTTATTTCTCAAAAGCCAGTCTTGCTCTGGTTTCTGGATTAAAGAGAGAGGGTGAGTGCCTTGCCCACTGTGGTCATGGATGCAAGATATTCACAGAAAATTAGCATCATAGAAAAAAAANNNAAAAAAAAAAAAAAAAAANCATGTCGGCCGCCTCGGCCAAACATCGGGTCGAGCATGCATCTAGGGCGGCCAATTCCGCCCCTCTCCCCCCCNGCNNTTTCCACACCGAGGAGTCCAGTTTGTGCAAGACACCCAGCGGAACCAAAACCCATCGTGGTATGTGAATCGAAGTCATCATAAAAGGTGACCCTTCTGTCTGTAAGATTTTAATTTAAGCATATATTTATGACCTCAACAAAGACGAACCATCTTTTGTTAATTCACCGTAGTAACACATAAAGTTATGCTACCTCTGATCAAAG-3′ (SEQ ID NO: 23).

Example 3 Additional Cloning Techniques

[0101] A nucleic acid molecule encoding a mu3 or mu4 polypeptide iscloned using a human testis Creator SMART cDNA library constructed inpDNR-LIB, a Creator donor vector. This vector has a probability ofgreater than 93 percent of obtaining a full-length cDNA. Once obtained,the full-length cDNA is sequenced and cloned into an expression vectorsuch as pCMV-Sport-bgal (Life Technologies). The expression vectorcontaining the nucleic acid encoding a mu3 or mu4 polypeptide istransfected into mammalian cells (e.g., CHO or Cos7) by, for example, byLipofection. Once transfected, the mammalian cells are analyzed formorphine and opioid peptide binding as well as naloxone sensitivity.

[0102] In addition, mRNA expression of a mu3 or mu4 opiate receptor isanalyzed by RT-PCR using real time PCR (GeneAmp 5700 sequence detection;Applied Biosystems) or by Northern blot analysis using asequence-specific probe as described herein.

[0103] The following procedures are performed to express nucleic acidencoding a polypeptide having opioid receptor activity. Briefly, a mu3or mu4 cDNA obtained from the library is cloned into apcDNA5/FRT/TO-TOPO expression vector (pcDNA5/FRT/TO TA Expression Kit,Invitrogen). This 5.2 kb expression vector is designed to facilitaterapid cloning and tetracycline-regulated expression of PCR productsusing the Flp-In T-REX System. The expression vector containing the geneof interest is cotransfected with the pOG44 Flp recombinase expressionplasmid into a Flp-In T-REX mammalian host cell line (Flp-In CHO) bylipid mediated transfection (Invitrogen), and the pcDNA5/FRT/TO-TOPOvector plus the DNA insert is integrated in a Flp recombinase-dependentmanner into the genome. Addition of tetracycline to the culture mediumcauses expression of the polypeptide encoded by the insert. ThepcDNA5/FRT/TO-TOPO expression vector is controlled by the strong humanCMV immediate early enhancer/promoter into which the tet operator2(TetO2) sequence have been inserted in tandem. Insertion of these TetO2sequences into the CMV promoter confers regulation by tetracycline tothe promoter. PCR primers are designed to ensure that the rightrecombinant protein is obtained. A pcDNA5/FRT/TO/CAT positive controlvector and a mock transfection (negative control) is used to evaluatethe results. The CAT protein expressed from the positive control plasmidis determined by ELISA or Western blot assays. Human mu3 or mu4 opiatereceptor polypeptide expression is determine by Western blot usingpolyclonal antibodies specific for either the human mu3 or mu4polypeptides. Polyclonal antibody that recognize these polypeptides aregenerated commercially. After identifying cells expressing the mu3 ormu4 polypeptide, functions such as the ability of morphine to cause theinduction of cNOS is evaluated. In addition, mu antagonists such asnaloxone and CTOP are used in addition to the NO synthase inhibitor,L-NAME, to evaluate the activity of the mu3 or mu4 polypeptide.

Example 4 Detecting Mu4 mRNA Expression

[0104] Human heparinized whole blood cells obtained from volunteer blooddonors (Long Island Blood Services; Melville, N.Y.) were immediatelyseparated using the 1-Step Polymorphs (Accurate Chemical and ScientificCorporation, Westbury, N.Y.) gradient medium. Five mL of the heparinizedblood was layered over 3.5 mL of polymorphs in a 14 mL round-bottom tubeand centrifuged for 35 minutes at 500×g in a swinging-bucket rotor at18° C. After centrifugation, the top band at the sample/medium interfaceconsisting of mononuclear cells was harvested in 14 mL tubes and thenwashed with RPMI 1640 media (GIBCO BRL, Gaithesburg, Md.) bycentrifugation for 10 minutes at 400×g. In addition, residual red bloodcells were lysed using ACK lysing buffer (Current Protocol inImmunology). The mononuculear cells were incubated in RPMI 1640supplemented with 10% fetal calf serum for 4 hours in a 37° C. incubatorwith 5% CO₂ in order to recover. The cells were then treated with SNAP(1 μM), SNAP plus superoxide dismutase (SOD; 100 units/mL) (SIGMA St.Louis, Mo.), or SOD (100 U/mL), respectively.

[0105] After incubation, mononuclear cells were pelleted bycentrifugation, and total RNA was isolated with the RNeasy Protect MiniKit (Qiagen, Stanford, Calif.) following the directions supplied by themanufacturer. RNA was eluted with 50 μL of RNase-free water.

[0106] First strand cDNA synthesis was performed using random hexamers(GIBCO, BRL, Gaithesburg, Md.). 3 μg of total RNA isolated from humanmononuclear cells were denatured at 95° C. and reverse transcribed at40° C. for 1 hour using Superscript II Rnase H-RT (GIBCO BRL,Gaithesburg Md.). Five μL of the RT product was used for the real-timePCR reaction.

[0107] Primers and probe specific for the mu4 opiate receptor sequencewere designed as follows using the software Primer Express (AppliedBiosystems). The sequence for the forward primer was5′-GAATCCTTTTCATTTTCCCCAGAAT-3′ (SEQ ID NO: 24); the sequence for thereverse primer was 5′-AACCAGAGCAAGACTGGCTTTTG-3′ (SEQ ID NO: 25); andthe sequence for the Taqman probe was5′-ATAATTCATAGATGTTGCTGCAATACCCCTCTTATTTCT-3′ (SEQ ID NO: 26). TheTaqman probe was constructed with the 5′ reporter dye6-carboxyfluorescein and a 3′ quencher dye6-carboxy-tetramethyl-rhodoamine. The 2× universal master mix (AppliedBiosystems) containing PCR buffer, MgCl₂, dNTPs, and the thermal stableAmpliTaq Gold DNA polymerase was used in the PCR reactions. In addition,200 μM of reverse and forward primers, 100 μM Taqman probe, 5 μL of RTproduct, and Rnase/DNase-free water were added to the master mix to afinal volume of 50 μL. The PCR reaction mixture was transferred to aMicroAmp optical 96-well reaction plate and incubated at 95° C. for 10minutes to activate the Amplitaq Gold DNA polymerase. The reactions wereperformed with 40 cycles at 95° C. for 30 seconds and 60° C. for 1minute on the Applied Biosystems GeneAmp 5700 Sequence Detection System.The PCR results were analyzed with the GeneAmp 5700 SDS software(Applied Biosystems). In order to determine the relative copy number ofthe target gene transcript, control cDNA generated from SHY cell totalRNA was used to produce a standard curve. A standard curve for thereference gene β-actin was performed using the Applied Biosystemsβ-actin TaqMan Control Reagents kit (part no. 401846). Viability countswere done for all of the different time points and 96% of themononuclear cells were viable.

[0108] Real-time RT-PCR analysis of human mononuclear cells treated withSNAP alone for 30 minutes resulted in significantly lower mu4 mRNAexpression (0.05 relative mRNA level) as compared to nontreated cells(1.0 relative mRNA level), whereas cells treated with SNAP plus SOD(which scavenges free radicals) for the same time period exhibited alevel of mu4 mRNA expression (0.83 relative mRNA level) close to theobserved control level (FIG. 2). After 6 hours of treatment, the levelof mu4 mRNA expression in cells treated with SNAP not only reboundedback but also significantly (p=0.029) exceeded control levels (8±5.6relative mRNA level; FIG. 3). After 24 hours of treatment, the level ofmu4 mRNA expression in cells treated with SNAP also exceeded controllevels (n=1; 10.5 relative mRNA level; FIG. 3). Similar results wereobserved in human mononuclear cells treated with morphine (1 μM; FIG.4).

OTHER EMBODIMENTS

[0109] It is to be understood that while the invention has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention, which is defined by the scope of the appended claims.Other aspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. An isolated nucleic acid molecule that encodes apolypeptide having mu3 opiate receptor activity.
 2. The isolated nucleicacid molecule of claim 1, wherein said isolated nucleic acid moleculecomprises a nucleic acid sequence with a length and a percent identityto the sequence set forth in SEQ ID NO: 1 over said length, wherein thepoint defined by said length and said percent identity is within thearea defined by points A, B, C, and D of FIG. 1, wherein point A hascoordinates (81, 100), point B has coordinates (81, 65), point C hascoordinates (15, 65), and point D has coordinates (15, 100).
 3. Theisolated nucleic acid molecule of claim 1, wherein said polypeptidecomprises an amino acid sequence with a length and a percent identity tothe sequence set forth in SEQ ID NO: 2 over said length, wherein thepoint defined by said length and said percent identity is within thearea defined by points A, B, C, and D of FIG. 1, wherein point A hascoordinates (26, 100), point B has coordinates (26, 65), point C hascoordinates (5, 65), and point D has coordinates (5, 100).
 4. Theisolated nucleic acid molecule of claim 1, wherein said isolated nucleicacid molecule hybridizes under hybridization conditions to the sense orantisense strand of the sequence set forth in SEQ ID NO: 1 or
 3. 5. Theisolated nucleic acid molecule of claim 1, wherein said isolated nucleicacid molecule comprises the sequence set forth in SEQ ID NO: 4, 6, 8, or10.
 6. An isolated nucleic acid molecule that hybridizes underhybridization conditions to the sense or antisense strand of a nucleicacid that encodes a polypeptide having mu3 opiate receptor activity,wherein said isolated nucleic acid molecule is at least 12 nucleotidesin length, and wherein said isolated nucleic acid molecule does nothybridize to the sense or antisense strand of the sequence set forth inSEQ ID NO: 12 or
 13. 7. An isolated nucleic acid molecule comprising anucleic acid sequence with a length and a percent identity to thesequence set forth in SEQ ID NO: 1 over said length, wherein the pointdefined by said length and said percent identity is within the areadefined by points A, B, C, and D of FIG. 1, wherein point A hascoordinates (81, 100), point B has coordinates (81, 65), point C hascoordinates (15, 65), and point D has coordinates (15, 100).
 8. Theisolated nucleic acid molecule of claim 7, wherein said isolated nucleicacid molecule encodes a polypeptide having mu3 opiate receptor activity.9. A cell comprising an isolated nucleic acid molecule that encodes apolypeptide having mu3 opiate receptor activity.
 10. The cell of claim9, wherein said isolated nucleic acid molecule comprises a nucleic acidsequence with a length and a percent identity to the sequence set forthin SEQ ID NO: 1 over said length, wherein the point defined by saidlength and said percent identity is within the area defined by points A,B, C, and D of FIG. 1, wherein point A has coordinates (81, 100), pointB has coordinates (81, 65), point C has coordinates (15, 65), and pointD has coordinates (15, 100).
 11. The cell of claim 9, wherein saidpolypeptide comprises an amino acid sequence with a length and a percentidentity to the sequence set forth in SEQ ID NO: 2 over said length,wherein the point defined by said length and said percent identity iswithin the area defined by points A, B, C, and D of FIG. 1, whereinpoint A has coordinates (26, 100), point B has coordinates (26, 65),point C has coordinates (5, 65), and point D has coordinates (5, 100).12. The cell of claim 9, wherein said isolated nucleic acid moleculehybridizes under hybridization conditions to the sense or antisensestrand of the sequence set forth in SEQ ID NO: 1 or
 3. 13. The cell ofclaim 9, wherein said isolated nucleic acid molecule comprises thesequence set forth in SEQ ID NO: 4, 6, 8, or
 10. 14. A cell comprisingan isolated nucleic acid molecule that hybridizes under hybridizationconditions to the sense or antisense strand of a nucleic acid thatencodes a polypeptide having mu3 opiate receptor activity, wherein saidisolated nucleic acid molecule is at least 12 nucleotides in length, andwherein said isolated nucleic acid molecule does not hybridize to thesense or antisense strand of the sequence set forth in SEQ ID NO: 12 or13.
 15. A substantially pure polypeptide having mu3 opiate receptoractivity.
 16. The substantially pure polypeptide of claim 15, whereinsaid polypeptide is encoded by a nucleic acid sequence having a lengthand a percent identity to the sequence set forth in SEQ ID NO: 1 oversaid length, wherein the point defined by said length and said percentidentity is within the area defined by points A, B, C, and D of FIG. 1,wherein point A has coordinates (81, 100), point B has coordinates (81,65), point C has coordinates (15, 65), and point D has coordinates (15,100).
 17. The substantially pure polypeptide of claim 15, wherein saidpolypeptide comprises an amino acid sequence with a length and a percentidentity to the sequence set forth in SEQ ID NO: 2 over said length,wherein the point defined by said length and said percent identity iswithin the area defined by points A, B, C, and D of FIG. 1, whereinpoint A has coordinates (26, 100), point B has coordinates (26, 65),point C has coordinates (5, 65), and point D has coordinates (5, 100).18. The substantially pure polypeptide of claim 15, wherein saidpolypeptide is encoded by a nucleic acid molecule that hybridizes underhybridization conditions to the sense or antisense strand of thesequence set forth in SEQ ID NO: 1 or
 3. 19. The substantially purepolypeptide of claim 15, wherein said polypeptide comprises the sequenceset forth in SEQ ID NO: 5, 7, 9, or
 11. 20. A method for identifying amu3 opiate receptor agonist, said method comprising: a) contacting acell with a test molecule, wherein said cell comprises an isolatednucleic acid molecule that encodes a polypeptide having mu3 opiatereceptor activity, and wherein said cell expresses said polypeptide, andb) determining whether or not said test molecule induces, in said cell,a mu3 opiate receptor-mediated response.
 21. The method of claim 20,wherein said determining step comprises monitoring nitric oxide synthaseactivity in said cell.
 22. The method of claim 21, wherein saidmonitoring nitric oxide synthase activity comprises detecting nitricoxide release from said cell.
 23. The method of claim 22, wherein anitric oxide-specific amperometric probe is used to detect said nitricoxide release.
 24. The method of claim 20, wherein said determining stepcomprises monitoring intracellular calcium levels within said cell. 25.The method of claim 24, wherein a fluorescent ion indicator is used tomonitor said intracellular calcium levels.
 26. The method of claim 25,wherein said fluorescent ion indicator comprises Fura-2.
 27. The methodof claim 20, wherein said determining step comprises monitoring nitricoxide synthase activity and intracellular calcium levels in said cell.28. A method for identifying a mu3 opiate receptor antagonist, saidmethod comprising: a) contacting a cell with a test molecule and a mu3opiate receptor agonist, wherein said cell comprises an isolated nucleicacid molecule that encodes a polypeptide having mu3 opiate receptoractivity, and wherein said cell expresses said polypeptide, and b)determining whether or not said test molecule reduces or prevents, insaid cell, a mu3 opiate receptor-mediated response induced by said mu3opiate receptor agonist.
 29. The method of claim 28, wherein said mu3opiate receptor agonist comprises morphine or dihydromorphine.
 30. Themethod of claim 28, wherein said determining step comprises monitoringnitric oxide synthase activity in said cell.
 31. The method of claim 28,wherein said determining step comprises monitoring intracellular calciumlevels within said cell.
 32. An isolated nucleic acid moleculecomprising a nucleic acid sequence with a length and a percent identityto the sequence set forth in SEQ ID NO: 22 over said length, wherein thepoint defined by said length and said percent identity is within thearea defined by points A, B, C, and D of FIG. 1, wherein point A hascoordinates (225, 100), point B has coordinates (225, 65), point C hascoordinates (15, 65), and point D has coordinates (15, 100).